Protein belonging to the TNF superfamily involved in signal transduction, nucleic acids encoding same and methods of use thereof

ABSTRACT

A method of modulating immune response in an animal is disclosed. Such a method interacting the immature dendritic cells from the animal with an antigen ex vivo so that the immature dendritic cells present the antigen on their surfaces, inducing maturation of the immature dendritic cells ex vivo, and contacting the mature dendritic cells ex vivo with a modulator comprising TRANCE, conservative variants thereof, fragments thereof, analogs or derivatives thereof, or a fusion protein comprising the amino acid sequence of TRANCE, conservative variants thereof, or fragments thereof. After contacting the modulator ex vivo, the mature dendritic cells are introduced into the animal. As a result, immune response in the animal towards the antigen is modulated relative to the immune response against the antigen in an animal in which dendritic cells did not interact with the antigen ex vivo, and did not contact a modulator ex vivo. Preferably, the method of the present invention results in increasing immune response towards the antigen in the animal.

DOMESTIC PRIORITY CLAIM

The priority is claimed of U.S. Provisional Application No. 60/069,589filed on Dec. 12, 1997, which is hereby incorporated by reference hereinin its entirety.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/595,524, filed Nov. 9, 2006, which is a continuation of U.S.application Ser. No. 11/032,797, filed Jan. 11, 2005, now U.S. Pat. No.7,393,927, which is a divisional of U.S. application Ser. No.09/873,829, filed May 9, 2002, now U.S. Pat. No. 7,063,960, which is aContinuation-In-Part of U.S. application Ser. No. 09/210,115, filed Dec.11, 1998, now abandoned, which is a Continuation-In-Part of U.S.application Ser. No. 09/034,099, filed Mar. 3, 1998, now abandoned,which is a Continuation-In-Part of U.S. application Ser. No. 08/989,479,now abandoned, and U.S. Provisional Patent Application 60/069,589, bothfiled Dec. 12, 1997, all of which are herein incorporated by referencein their entireties.

GOVERNMENT RIGHTS CLAUSE

The research leading to the present invention was supported in part withNational Institutes of Health MSTP Grant GM07739 and National Institutesof Health Grant Nos: CA525133, AI13013 and AI13672. The government mayhave rights in the invention.

FIELD OF INVENTION

The present invention relates to the identification and characterizationof a protein belonging to the TNF superfamily which is involved insignal transduction, nucleic acids which encode the protein, and usesfor the nucleic acids, the protein, and products derived therefrom, suchas antibodies and pharmaceutical compositions. In particular, theprotein of the present invention is involved in the modulation of thesurvivability of mature dendritic cells of the immune system. As aresult, the present invention has numerous uses including, but notlimited to, modulating immune response to an antigen, diagnosing immunesystem related conditions, and treating immune system relatedconditions.

BACKGROUND OF THE INVENTION

Mature drendritic cells are specialized cells that play a role in immuneresponse. They develop from pluripotent hemopoietic stem cells locatedin bone marrow, and function to present antigens on their surface inorder to activate T cells and generate an immune response to aparticular antigen. Major Histocompatibility Complex (MHC) proteins,such as Class I MHC molecules and Class II MHC molecules, are, involvedin the presentation of an antigen on the surface of a mature dendriticcell. The activation of T cells involves a costimulatory process. Onesignal is from the antigen bound to the MHC molecule on the surface ofthe mature dendritic cell. This complex interacts with the T cellreceptor complex on the surface of the T cell. The other signal resultsfrom molecules produced by the mature dendritic cell, which bind toreceptors on the T cell. The T cell becomes activated upon receivingboth signals, and undergoes an autocrine process wherein it separatesfrom the mature dendritic cell and simultaneously secretes a growthfactor like IL-2 along with cell-surface receptors that bind to it. Thebinding of IL-2 to its receptor stimulates the T cell to proliferate, solong as it has already encountered its specific antigen.

Once the T cell disengages from the mature dendritic cell, another Tcell can bind the MHC-antigen complex on the surface of the maturedendritic cell, and be activated. Hence, the longer an antigenpresenting mature dendritic cell can survive, the greater the number ofT cells it can activate, and the immune response to the specific antigenwill be more efficient. However, pluripotent hematopoietic stem cellsare constantly undergoing differentiation, and new dendritic cells areconstantly being produced. In order to maintain and develop the immunesystem, mature dendritic cells ultimately undergo apoptosis, wherein itsnucleus shrinks and condenses, and the cell shrivels and dies. Newlyproduced mature dendritic cells are constantly replacing these dead anddying mature dendritic cells.

Members of the tumor necrosis factor (TNF) superfamily can regulateapoptosis in addition to an array of other biological effects, such ascell proliferation, and differentiation. The TNF superfamily currentlyincludes TNF, LT-α, LT-β, FasL, CD40L, CD30L, CD27L, 4-1BBL, OX40L (1)and TRAIL/APO-2L (2, 3) which exhibit the highest homology between theirC-terminal, receptor binding domains. The superfamily members are typeII membrane proteins that act in an autocrine, paracrine or endocrinemanner either as integral membrane proteins or as proteolyticallyprocessed soluble effectors. Despite the functional redundancy of thisfamily, specificity may be accomplished by coordinating the spatial andtemporal expression of TNF-related ligands and their receptors, and byrestricting the expression of signal transduction molecules to specificcell types. TNF receptors interact with a family of molecules calledTRAFs (TNF receptor associated proteins) that act as adaptors for thedownstream signaling events. Hence, binding of a TNF cytokine to itscognate receptor, which is interacting with TRAF, leads to theactivation of several signal transduction pathways, including theactivation of the cascade of caspase/ICE-like proteases, which areresponsible for apoptosis. Also activated is the nuclear factor-κB(NF-κB) family of transcription factors, which inhibit apoptosis, andmitogen activated protein kinases including the c-Jun N-terminal proteinkinases (JNK) and the extracellularly-regulated kinases (ERK).

Moreover, the TNF receptor family can also regulate apoptosis bymodulating the expression of the proto-oncogene bcl-2 to produce Bcl-2and Bcl-2 related proteins. Bcl-2 can suppress apoptosis in the cell byaltering transmembrane conductance in mitochondria and preventing theactivation of the caspase/ICE-like proteases.

As explained above, the longer an antigen presenting mature dendriticcell can survive, the greater the number of T cells it can activate, andthe immune response to the specific antigen will be more efficient.Accordingly, there is a need to be able to increase the active life ofantigen presenting mature dendritic cells, and to inhibit apoptosis insuch cells.

There is a further need to exploit the increased survivability ofantigen presenting dendritic cells to modulate the immune response to anantigen. For example, such increased survivability can be used todiagnose and treat immune system related conditions.

Such increases in mature dendritic cell survivability can also be usedto modulate T cell activation in an animal, and thereby modulate theimmune response to an antigen.

The citation of any reference herein should not be construed as anadmission of such reference as prior art.

SUMMARY OF THE INVENTION

The present invention relates to a novel TNF superfamily member membranebound protein designated TNF-Related Activation Induced Cytokine,hereinafter referred to as “TRANCE.” TRANCE is selectively expressed onT cells, and its receptor, hereinafter referred to as “TRANCE-R”, hasbeen detected on the surface of mature dendritic cells located inlymphoid tissues, such as the lymph node and thymus. It has beendetermined that the interaction of TRANCE with TRANCE-R on the surfaceof a mature dendritic cell results in the upregulation of the expressionby the cell of the Bcl-x_(L) protein. This protein, related to Bcl-2,suppresses apoptosis in the cell by altering transmembrane conductancein mitochondria and preventing the activation of the caspase/ICE-likeproteases. Hence, the exposure of an a mature dendritic cell to TRANCEwill increase its life span.

Thus, in a first embodiment, the present invention relates to anisolated nucleic acid molecule comprising the DNA sequence set forth inFIG. 1 (SEQ ID NO:1), or degenerate variants thereof, which correspondto the human TRANCE gene, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof. Moreover, this embodimentextends to an isolated nucleic acid molecule hybridizable to theisolated nucleic acid molecule of FIG. 1 (SEQ ID NO:1), or degeneratevariants thereof, under standard hybridization conditions. Yet further,the invention includes isolated nucleic acid molecules that encodepolypeptides having an amino acid sequence as set forth in FIG. 2 (SEQID NO:2), which is the amino acid sequence of human TRANCE, as well asconservative variants thereof.

In another embodiment, the present invention relates to an isolatednucleic acid molecule comprising the DNA sequence set forth in FIG. 3(SEQ ID NO: 3), degenerate variants thereof, which correspond to themurine TRANCE gene, degenerate variants thereof, fragments thereof, oranalogs or derivatives thereof. Furthermore, this embodiment alsoincludes an isolated nucleic acid molecule hybridizable to the nucleicacid molecule of FIG. 3 (SEQ ID NO:3), or degenerate variants thereof,under standard hybridization conditions. Likewise included are nucleicacids molecules that encode the murine TRANCE protein, having an aminoacid sequence as set forth in FIG. 4 (SEQ ID NO:4), as well asconservative variants thereof.

Naturally, the present invention extends to the amino acid sequences ofhuman and murine TRANCE, conservative variants thereof, and fragments ofhuman and murine TRANCE, and conservative variants thereof, wherein thehuman TRANCE corresponds to the amino acid sequence as set forth in FIG.2 (SEQ ID NO:2), and the murine TRANCE corresponds to the amino acidsequence as set forth in FIG. 4 (SEQ ID NO:4).

Also included in the present invention are detectably labeled nucleicacids hybridizable to an isolated nucleic acid having a DNA sequence asset forth in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3), degeneratevariants thereof or fragments thereof.

In addition, the present invention also includes antibodies whereinTRANCE, conservative variants thereof, or a fragment thereof, is theimmunogen used in production of the antibodies. These antibodies can bemonoclonal or polyclonal. Moreover, the antibodies can be “chimeric” as,for example, they may comprise protein domains from anti-TRANCEantibodies raised against TRANCE in different species.

Moreover, the present invention includes an antibody of TRANCEdetectably labeled so that its bonding to TRANCE can be detected. Suchdetectable labels include enzymes conjugated to the antibody, such asalkaline phosphatase or peroxidase, or radioactive isotopes incorporatedinto the structure of the antibody.

A further embodiment of the present invention extends to an expressionvector containing a nucleic acid molecule which encodes TRANCE,degenerate variants or fragments the isolated nucleic acid molecule, oran isolated nucleic acid hybridizable to the nucleic acid molecule whichencodes TRANCE or degenerate variants thereof, under standardhybridization conditions, operatively associated with a promoter. Withthis expression vector, one may transfect or transform a unicellularhost which can then produce TRANCE. The method for expressing TRANCEcomprises introducing an expression vector of the present invention intoa host cell in a culture to cause a unicellular host into which thevector is introduced, to express an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NOS: 1 or 3, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof, andthereby produce TRANCE, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof, and then recover the TRANCEfrom the unicellular host, the culture, or both. In one embodiment, anexpression vector may comprise the isolated nucleic acid moleculecomprising the DNA sequence set forth in FIG. 1 (SEQ ID NO:1),degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, operatively associated with a promoter. In anotherembodiment, the expression vector comprises the nucleic acid moleculecomprising the DNA sequence of FIG. 3 (SEQ ID NO:3), degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof,operatively associated with a promoter. An expression vector can beintroduced into numerous types of unicellular hosts in order to produceTRANCE, including, but not limited to mammalian cells, insect cells, andbacterial cells.

An expression vector of the present invention can employ numerouspromoters to express TRANCE. The promoters applicable to the presentinvention, include, but are not limited to early promoters of hCMV,early promoters of SV40, early promoters of adenovirus, early promotersof vaccinia, early promoters of polyoma, late promoters of SV40, latepromoters of adenovirus, late promoters of vaccinia, late promoters ofpolyoma, the lac the trp system, the TAC system, the TRC system, themajor operator and promoter regions of phage lambda, control regions offd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatasepromoter, or promoters of yeast a mating factor.

As explained above, unicellular hosts can be transformed with anexpression of an isolated nucleic acid molecule of the invention. Suchunicellular hosts include, but are not limited to, E. coli, Pseudonomas,Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1,BSC40, BMT10 and Sf9 cells. Moreover, mammalian cells can be used as aunicellular host.

The present invention also includes a mammalian cell containing anisolated nucleic acid encoding TRANCE, examples of which are describedherein, wherein the isolated nucleic acid sequence is modified in vitroto permit higher expression of a TRANCE polypeptide in the cell by meansof a homologous recombinational event, which comprises inserting anexpression regulatory sequence, such as a promoter, in functionalproximity to the TRANCE polypeptide encoding sequence. In such a cell,the expression regulatory sequence can be a TRANCE promoter and thehomologous recombinational event replaces a mutant TRANCE promoter.However, numerous promoters described herein can also be inserted insuch a cell and result in higher expression of TRANCE.

Also included in the present invention are nucleic acid fragments of theisolated nucleic acid molecule of the present invention, degeneratevariants thereof, and isolated nucleic acid molecules which hybridize tosuch fragments or degenerate variants thereof, under standardhybridization conditions. Such fragments may also be inserted intoexpression vectors and operatively associated with a promoter, which inturn, can be used to cause a host cell in which the vector is introducedto express and thereby produce such fragments of TRANCE.

Accordingly, a method for producing TRANCE fragments is included in thepresent invention, wherein this method comprises the steps ofintroducing an expression vector of the present invention containing afragment of an isolated nucleic acid molecule encoding TRANCEoperatively associated with a promoter into a unicellular host in aculture to cause the host cell into which the expression vector isintroduced to express the isolated nucleic acid molecule and therebyproduce a TRANCE, a conservative variant thereof, a fragment thereof, oranalog or derivative thereof, and then recovering the TRANCE from theunicellular host, its culture, or both. Examples of unicellular hostcells which can be used in this embodiment of the present invention aredescribed above. In one embodiment, the nucleic acid fragment is fromthe isolated nucleic acid molecule comprising a DNA sequence as setforth in FIG. 1 (SEQ ID NO:1), and the nucleic acid fragment encodes anactive peptide fragment of TRANCE. In another embodiment, the nucleicacid fragment is from the isolated nucleic acid molecule comprising aDNA sequence as forth in FIG. 3 (SEQ ID NO:3), and the nucleic acidfragment encodes an active peptide fragment of TRANCE. The expressionvectors of the present invention can be successfully introduced intonumerous types of host cells in order to produce TRANCE fragments,including, but not limited to, mammalian cells, insect cells, andbacterial cells.

The present invention further comprises a modulator of immune responsein a mammal. The modulator can be a polypeptide having an amino acidsequence as set forth in FIG. 2 (SEQ ID NO:2), conservative variantsthereof or a fragment thereof, or a polypeptide having an amino acidsequence as set forth in FIG. 4 (SEQ ID NO:4), conservative variantsthereof or a fragment thereof. The modulator can also be an analog orderivative of a polypeptide having an amino acid sequence as set forthin FIG. 2 (SEQ ID NO:2), conservative variants thereof or a fragmentthereof, or an analog or derivative of a polypeptide having an aminoacid residue sequence as set forth in FIG. 4 (SEQ ID NO:4), orconservative variants thereof, or a fragment thereof. Moreover, themodulator can be a fusion protein comprising an amino acid sequence asset forth in FIG. 2 (SEQ ID NO:2), conservative variants thereof or afragment thereof, or an amino acid sequence as set forth in FIG. 4 (SEQID NO:4), conservative variants thereof, or a fragment thereof. Otherforms of the modulator include an anti-sense TRANCE nucleic acidcomprising at least one phosphodiester analog bond, and an antibody,wherein its immunogen is selected from the group consisting of apolypeptide having an amino acid sequence as set forth in FIG. 2 (SEQ IDNO:2), conservative variants thereof, or a fragment thereof, apolypeptide having an amino acid sequence as set forth in FIG. 4 (SEQ IDNO:4), conservative variants thereof, or a fragment thereof, an analogor derivative of a polypeptide having an amino acid sequence as setforth in FIG. 2 (SEQ ID NO:2), conservative variants thereof, or afragment thereof, an analog or derivative of a polypeptide having anamino acid sequence as set forth in FIG. 4 (SEQ ID NO:4), conservativevariants thereof or a fragment thereof; a fusion protein wherein itsamino acid sequence comprises an amino acid sequence as set forth inFIG. 2 (SEQ ID NO:2), conservative variants thereof; or a fragmentthereof; and a fusion protein wherein its amino acid sequence comprisesthe amino acid sequence set forth FIG. 4 (SEQ ID NO:4), conservativevariants thereof, or a fragment thereof.

Moreover, also included in the present invention are analogs orderivatives of TRANCE, wherein a water soluble polymer is conjugated toa TRANCE protein. An example of such a polymer is polyethylene glycol.Moreover, an analog or derivative of a TRANCE protein can be mono-, di-,tri- or tetrapegylated. Moreover, pegylation of TRANCE to from a TRANCEanalog or derivative can occur at the N terminus, such that TRANCE isN-terminal monopegylated.

The function of the modulators is to modulate the life span of maturedendritic cells, and hence modulate immune response in the mammal. Inparticular, exposure of mature dendritic cells to a modulator which isan agonist of TRANCE, such as a polypeptide having an amino acidsequence as set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof or a fragment thereof; an analog orderivative of a polypeptide having an amino acid sequence as set forthin FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservative variantsthereof or a fragment thereof; a fusion protein wherein its amino acidsequence comprises the amino acid sequence of FIG. 2 (SEQ ID NO:2), FIG.4 (SEQ ID NO:4), or conservative variants thereof or a fragment thereof;causes the upregulation of expression of Bcl-x_(L), which inhibitsapoptosis. As a result, mature dendritic cells have a greater activelife span than mature dendritic cells not exposed to TRANCE or anagonist modulator thereof, and hence can activate a greater number of Tcells than are activated by mature dendritic cells not exposed to aTRANCE agonist modulator.

Also disclosed are TRANCE antagonist modulators which function todecrease the active life span of mature dendritic cells. In particular,these modulators can form a complex with TRANCE on the surface of Tcells and prohibit TRANCE from interacting with TRANCE-R. TRANCEantagonist modulators can also form a complex with TRANCE mRNA, andprevent its translation. As a result, the signal transduction of TRANCEis blocked, the upregulation of the expression of Bcl-x_(L) in maturedendritic cells does not occur, and apoptosis of the cell is notinhibited. Hence, these mature dendritic cells activate less T cellsthan are activated by mature dendritic cells exposed to TRANCE, and theimmune response in the mammal is decreased. The TRANCE antagonistmodulators comprise an anti-sense TRANCE nucleic acid comprising atleast one phosphodiester analog bond, or an antibody, wherein itsimmunogen is selected from the group consisting of a polypeptide havingan amino acid sequence as set forth in FIG. 2 (SEQ ID NO:2),conservative variants thereof, or fragment thereof, a polypeptide havingan amino acid sequence as set forth in FIG. 4 (SEQ ID NO:4),conservative variants thereof or a fragment thereof, an analog orderivative of a polypeptide having an amino acid sequence as set forthin FIG. 2 (SEQ ID NO:2), conservative variants thereof or a fragmentthereof, an analog or derivative of a polypeptide having an amino acidsequence as set forth in FIG. 4 (SEQ ID NO:4), conservative variantsthereof or a fragment thereof, a fusion protein wherein its amino acidsequence comprises the amino acid sequence of FIG. 2 (SEQ ID NO:2),conservative variants thereof or a fragment thereof, and a fusionprotein containing the amino acid sequence of FIG. 4 (SEQ ID NO:4),conservative variants thereof, or a fragment thereof.

The present invention also comprises a TRANCE agonist pharmaceuticalcomposition comprising a modulator which is an agonist of TRANCE, and apharmaceutically acceptable carrier thereof. The TRANCE agonistmodulator comprises a polypeptide comprising an amino acid sequence asset forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservativevariants thereof or a fragment thereof, an analog or derivative of apolypeptide having an amino acid sequence as set forth in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof or a fragmentthereof, a fusion protein wherein its amino acid sequence comprises theamino acid sequence of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), orconservative variants thereof or a fragment thereof.

As explained above, it has been determined that exposure of an antigenpresenting mature dendritic cell to TRANCE causes upregulation of theexpression of Bcl-x_(L), and inhibits apoptosis in a mature dendriticcell. Hence, a TRANCE agonist pharmaceutical composition can be used ina method for treating an immune system related condition in a mammal,wherein the method for treating an immune system related comprises thesteps of exposing at least one mature dendritic cell of the mammal to anantigen so that the at least one mature dendritic cell can present theantigen on its surface, and administering to the mammal atherapeutically effective amount of the TRANCE agonist pharmaceuticalcomposition. The exposure of the at least one mature dendritic cell tothe antigen can occur in vivo, wherein the antigen is administered to ananimal, or ex vivo, wherein the at least one mature dendritic cell isremoved from the mammal and exposed to an antigen in a medium whichpermits the presentation of the antigen on the surface of the at leastone mature dendritic cell. The at least one mature dendritic cellpresenting the antigen on its surface is then reintroduced into themammal prior to the administration of a TRANCE agonist pharmaceuticalcomposition. This method increases the active life of a mature dendriticcell presenting an the antigen on its surface. As a result, the numberof T cells activated against the antigen presented on the surface of themature dendritic cell is increased, which results in an increase in amammal's immune response to the antigen.

The antigen used in this method can be selected from the groupconsisting of a pathogen or a fragment thereof, a virus or a fragmentthereof, and a tumor, or a fragment thereof. The immune system relatedconditions treated with this method include, but are not limited toviruses which include, but are not limited to adenoviruses, and virusesrelated to cancer such as HIV, Papillomavirus, Hepatitis-B, Epstein-Barrvirus, and cancer itself.

Moreover, the present invention also includes a TRANCE antagonistpharmaceutical composition, which comprises the modulator which is anantagonist of TRANCE and a pharmaceutically acceptable carrier thereof.The modulator which is an antagonist of TRANCE is selected from thegroup consisting the anti-sense TRANCE nucleic acid comprising at leastone phosphodiester analog bond, or an antibody having an immunogenselected from the group consisting of the polypeptide having an aminoacid sequence as set forth in FIG. 2 (SEQ ID NO:2) or a fragmentthereof, the polypeptide having an amino acid sequence as set forth inFIG. 4 (SEQ ID NO:4) or a fragment thereof, an analog or derivative ofthe polypeptide having an amino acid sequence as set forth in FIG. 2(SEQ ID NO:2) or a fragment thereof, the analog or derivative of thepolypeptide having an amino acid sequence as set forth in FIG. 4 (SEQ IDNO:4) or a fragment thereof, the fusion protein containing the aminoacid sequence of FIG. 2 (SEQ ID NO:2), or a fragment thereof and thefusion protein containing the amino acid sequence of FIG. 4 (SEQ IDNO:4), or a fragment thereof.

Yet further provided in the present invention is a TRANCE antagonistpharmaceutical composition which modulates immune response in a mammalby preventing the upregulation of the expression of Bcl-x_(L) so thatapoptosis is not inhibited in mature dendritic cells. As a result, thelife span of mature dendritic cells of a mammal whose immune system isexposed to a TRANCE antagonist pharmaceutical composition is decreasedrelative to the life span of mature dendritic cells in a mammal whoseimmune system is not exposed to a TRANCE antagonist pharmaceuticalcomposition. The TRANCE antagonist pharmaceutical composition comprisesa modulator which is an antagonist of TRANCE, and is selected from thegroup consisting of an anti-sense TRANCE nucleic acid comprising atleast one phosphodiester analog bond, and an antibody, wherein theimmunogen of the antibody is selected from the group consisting of thepolypeptide having an amino acid sequence as set forth in FIG. 2 (SEQ IDNO:2) or a fragment thereof, the polypeptide having an amino acidsequence as set forth in FIG. 4 (SEQ ID NO:4) or a fragment thereof, theanalog or derivative of the polypeptide having an amino acid sequence asset forth in FIG. 2 (SEQ ID NO:2) or a fragment thereof, the analog orderivative of the polypeptide having an amino acid sequence as set forthin FIG. 4 (SEQ ID NO:4) or a fragment thereof, the fusion proteincontaining the amino acid sequence of FIG. 2 (SEQ ID NO:), or a fragmentthereof, and the fusion protein containing the amino acid sequence ofFIG. 4 (SEQ ID NO:4), or active fragment thereof, and a pharmaceuticallyacceptable carrier thereof.

Moreover, disclosed herein is a method for treating an immune systemrelated condition in a mammal with the TRANCE antagonist, pharmaceuticalcomposition, wherein the method comprises administering to the mammal atherapeutically effective amount of the TRANCE antagonist pharmaceuticalcomposition. As a result, the signal transduction of TRANCE from thesurface of a T cell is blocked, such that TRANCE can not interact withTRANCE-R, the upregulation of expression of Bcl-x_(L) in maturedendritic cells does not occur, and apoptosis is not inhibited. An aimmune system related condition treated with this method involvesoverexpression of TRANCE on the surface of T cells, and the life span ofmature dendritic cells is of such a length as to be detrimental to themammal. One example of such a condition is an autoimmune disease, suchas rheumatoid arthritis. Another example involves hypersensitivity to anallergen so that the mammal's immune response towards an allergen isless sever. As a result, this method of the present invention can beused in induce anergy in the mammal towards the allergen.

In yet another embodiment, the present invention can be used to diagnosean immune system related condition in a mammal, such as a human. Asdisclosed herein, TRANCE is a member of the TNF superfamily of proteins,and the exposure of mature dendritic cells to TRANCE ultimatelyincreases the life span of such cells. Hence, just as in the case ofother TNF receptor superfamily proteins such as CD40L, wherein its underexpression results in an immune system related condition called HyperIGM syndrome, the under expression of TRANCE can also result in animmune system related condition. Moreover, Autoimmune HumanLymphoproliferative Syndrome (ALPS) is a condition caused by a mutationin the TNF receptor superfamily protein Fas, such that the protein isnot expressed, or is expressed in a mutated form with either no activityor decreased activity relative to the wild type Fas protein.Consequently, the under expression or lack of expression of TRANCE in amammal results in an immune system related condition. Hence, the presentinvention provides a method for diagnosing such an immune system relatedcondition in a mammal such as a human, wherein the method comprises thesteps of removing a bodily sample from the mammal, assaying the bodilysample to determine whether TRANCE is expressed in the sample. Thebodily sample can be blood or lymphoid tissue such as lymph node tissue,spleen tissue or thymus tissue.

Such a lack of expression, or under expression, can result from anonsense mutation in the gene or cDNA encoding TRANCE whereby TRANCEmRNA is not translated, or it is mutated, and the protein productproduced from its translation is nonfunctional, or has decreasedfunction. Hence, the isolated nucleic acids of the present inventionwhich encode TRANCE, and the amino acid sequences of TRANCE disclosed inthe present invention, can be used for diagnosing an immune systemrelated condition in the mammal, such as autoimmune disease.

Antibodies to the modulators are also encompassed within the scope ofthe present invention. In particular, these antibodies can be monoclonalor polyclonal. Moreover, chimeric antibodies made against the modulatorfrom at least two species are included in the present invention.

Yet still another embodiment of the present invention is a method ofgene therapy for modulating levels of expression of a TRANCE protein ina mammal. Since T cells, like mature dendritic cells, originate frompluripotent hematopoietic stem cells, and are constantly being producedto replace cells which die from apoptosis, an alteration of the TRANCEgene in a pluripotent stem cell, or the addition of copies of the TRANCEgene operatively associated with a promoter to the genome of the stemcell will modulate the level of expression of TRANCE on the surface of Tcells. The method for accomplishing such modulation comprises the stepsof removing at least one hematopoietic stem cell from the mammal,destroying remaining hematopoietic stem cells in the mammal,transfecting the at least one hematopoietic stem cell with a vectorcontaining a nucleic acid molecule which encodes a TRANCE protein suchthat the nucleic acid molecule becomes incorporated into the genome ofthe hematopoietic stem cell forming a transfected hematopoietic stemcell, and introducing the transfected hematopoietic stem into the mammalso that the transfected hematopoietic stem cell can self replicate anddifferentiate within the mammal. In one embodiment, the nucleic acidwhich encodes TRANCE has a nucleotide sequence as set forth in FIG. 1(SEQ ID NO:1).

The present invention further extends to a method for modulating immuneresponse to an antigen in an animal so that the animal's immune systemcan effectively and efficiently destroy the antigen. More specifically,such a method of the present invention comprises the steps ofinteracting immature dendritic cells from the animal with an antigen exvivo, so that the cells present the antigen on their surfaces, inducingmaturation of the dendritic cells ex vivo, contacting the maturedendritic cells with a modulator of immune response ex vivo, andintroducing the mature dendritic cells into the animal. Contacting theantigen presenting mature dendritic cells with TRANCE or a TRANCEagonist ex vivo increases the in vivo survivability of the maturedendritic cells upon their introduction into the animal. As a result,the antigen presenting dendritic cells that have had ex vivo interactionwith the antigen, and contact with TRANCE or a TRANCE agonist activate agreater number of T cells in vivo, than can mature dendritic cells forwhich the method of the present invention was not performed.Consequently, the immune response towards the antigen in an animal forwhich the present invention was performed is modulated relative to theimmune response towards the antigen in an animal in which the method ofthe present invention was not performed. In a preferred embodiment, themethod for modulating immune response involves increasing the immuneresponse to the antigen in the animal. Preferably, the immaturedendritic cells of the animal comprise bone marrow derived immaturedendritic cells.

Furthermore, the present invention extends to numerous means ofinteracting immature dendritic cells with an antigen ex vivo, pursuantto a method of modulating immune response as set forth above. Forexample, immature dendritic cells can be transfected with an expressionvector comprising a nucleic acid which encodes the antigen, operativelyassociated with a promoter. Upon expression of the nucleic acidsequence, the antigen is produced within immature dendritic cells, andthen presented on their surfaces. All of the expression vectors andpromoters set forth above and having applications in the expression ofTRANCE, conservative variants thereof, fragments thereof, or analogs orderivatives thereof, have applications in this aspect of the invention.

In another example of interacting immature dendritic cells with theantigen, immature dendritic cells can be pulsed with the antigen exvivo. In this type of interaction, the antigen is taken up by theimmature dendritic cells, proteolytically processed therein, andpresented on the surface of the immature dendritic cells.

Moreover, examples of such antigens against which immune response can beincreased include pathogens, or fragments thereof, viruses or fragmentsthereof, or tumors, or viruses thereof, to name only a few.

Furthermore, examples of modulators having applications in a method formodulating immune response in an animal towards an antigen comprise:

-   -   a) a polypeptide having an amino acid sequence of FIG. 2 (SEQ ID        NO:2), FIG. 4 (SEQ NO:4), conservative variants thereof, or        fragments thereof;    -   b) an analog or derivative of a polypeptide having an amino acid        sequence set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID        NO:4), conservative variants thereof, or fragments thereof; or    -   c) a fusion protein having an amino acid sequence comprising an        amino acid sequence set forth in FIG. 2 (SEQ ID NO:2), FIG. 4        (SEQ ID NO:4), conservative variants thereof, or fragments        thereof.

Moreover, numerous methods, encompassed by the present invention, areavailable for introducing dendritic cells which had previouslyinteracted with the antigen, and contacted a TRANCE agonist, into ananimal. For example, such dendritic cells can be subcutaneously injectedinto the animal.

Naturally, methods of modulating immune response to an antigen in amammal as set forth above extends to mammals, and particularly, humans.

Furthermore, the present invention extends to a method for increasingthe viability of a dendritic cell, comprising contacting the dendriticcell with an isolated TRANCE comprising an amino acid sequence of SEQ IDNO:2, conservative variants thereof, fragments thereof, or analogs orderivatives thereof, wherein said dendritic cell is contacted by saidisolated TRANCE has an increased viability relative to control dendriticcell not contacted with said isolated TRANCE. In a particular embodimentof the invention, the dendritic cell is contacted with the TRANCEprotein while in an immature state. Also, such contact can occur invitro or in vivo.

Likewise, the present invention extends to a method of increasingviability of a dendritic cell, comprising contacting the dendritic cellwith an isolated TRANCE comprising an amino acid sequence of SEQ IDNO:4, conservative variants thereof, fragments thereof, or analogs orderivatives thereof, wherein said dendritic cell contacted by isolatedTRANCE has an increased viability relative to a control dendritic cellnot contacted with said isolated TRANCE, and such contact can occur invitro or in vivo.

Moreover, the present invention extends to a method of increasingviability of a dendritic cell, comprising pulsing the dendritic cellwith an isolated TRANCE comprising an amino acid sequence of SEQ IDNO:2, conservative variants thereof, fragments thereof, or analogs orderivatives thereof, and pulsing the dendritic cell with an isolatedprotein which is a member of the Tumor Necrosis Factor (TNF) superfamilyof proteins, such that the dendritic cell comprises an increasedviability relative to a control dendritic cell not pulsed with TRANCEand the protein. Optionally, the pulsing steps of the invention canoccur simultaneously.

In another embodiment, the invention comprises a method of increasingviability of a dendritic cell, comprising pulsing the dendritic cellwith isolated TRANCE comprising an amino acid sequence of SEQ ID NO:4,conservative variants thereof, fragments thereof, or analogs orderivatives thereof, and pulsing the dendritic cell with an isolatedprotein which is a member of the TNF superfamily of proteins, such thatthe pulsed dendritic cell comprises an increased viability relative to acontrol dendritic cell not pulsed with isolated TRANCE and the isolatedprotein. Also, such pulsing of the cell can occur simultaneously.

Numerous proteins well known to the skilled artisan are members of theTNF superfamily, which is explained and described throughout the instantSpecification. Particular examples of such proteins include, butcertainly are not limited to, CD40L or TNF-α.

The present invention further extends to a method for increasingviability of a dendritic cell of an animal in vivo, comprising:

-   -   Removing an immature dendritic cell from the animal;    -   pulsing the immature dendritic cell with an isolated TRANCE        comprising an amino acid sequence of SEQ ID NO:2, conservative        variants thereof, fragments thereof, or analogs or derivatives        thereof,    -   inducing the immature dendritic cell to mature; and    -   reintroducing the mature dendritic cell into the animal.

Optionally, the dendritic cells can be washed after the pulsing step,and prior to reintroducing them to the animal. Once in the animal, cellswhich have been treated in this matter will have an increased viability,i.e., a greater life span that dendritic cells which have not beentreated according to the teachings of the invention. Furthermore,various forms of TRANCE, including murine TRANCE comprising an aminoacid sequence of SEQ ID NO:4, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof, have applications in theinvention.

Furthermore, the instant invention also enables one of ordinary skill inthe art to increase an animal's immune response toward a particularantigen, and thus has applications in treating an immune system relatedcondition. In particular, the present invention extends to a method forincreasing immune response in an animal towards an antigen, comprisingthe steps of:

-   -   Removing an immature dendritic cell from the animal;    -   pulsing the immature dendritic cell with an isolated TRANCE        comprising an amino acid sequence of SEQ ID NO:2, conservative        variants thereof, fragments thereof, or analogs or derivatives        thereof;    -   pulsing the immature dendritic cell with the antigen;    -   inducing the immature dendritic cell to mature; and    -   reintroducing the mature dendritic cell into the animal.

Naturally, other TRANCE proteins, such as murine. TRANCE comprising anamino acid sequence of SEQ ID NO:4, conservative variants thereof,fragments thereof, or analogs or derivatives thereof, have applicationsherein.

In a particular embodiment of the invention, the dendritic cells whichhave been pulsed can be washed prior to reintroduction into the animal.

In addition, numerous antigens can be used in this embodiment of theinvention. Examples include, a pathogen, or a fragment thereof, a virus,or a fragment thereof or a tumor, or a fragment thereof. Hence, thisaspect of the invention can be readily used to treat an immune systemrelated condition, such as cancer of HIV, because it increases theanimal's immune response to an antigen that is associated with theimmune system related condition. Hence, such conditions can be readilytreated with the invention. Hence, naturally, antigens have applicationsherein include

-   -   a) a pathogen, or a fragment thereof;    -   b) a virus, or a fragment thereof; and    -   c) a tumor, or a fragment thereof.

Another embodiment involves Applicants' discovery of a synergisticcooperativity between TRANCE and a protein which is a member of the TNFsuperfamily in increasing the viability, e.g., the life span of adendritic cell. In particular, the present invention extends to a methodfor increasing immune response in an animal towards an antigen,comprising the steps of:

-   -   Removing an immature dendritic cell from the animal;    -   pulsing the immature dendritic cell with an isolated TRANCE        comprising an amino acid sequence of SEQ ID NO:2, conservative        variants thereof, fragments thereof, or analogs or derivatives        thereof;    -   pulsing the immature dendritic cell with an isolated protein of        the TNF superfamily;    -   pulsing the immature dendritic cell with the antigen;    -   inducing the immature dendritic cell to mature; and    -   reintroducing the mature dendritic cell into the animal.

As explained, above, numerous TRANCE proteins, including TRANCEcomprising an amino acid sequence of SEQ ID NO:4, conservative variantsthereof, fragments thereof, or analogs or derivatives thereof, haveapplications herein.

Moreover, numerous proteins of the TNF superfamily, which is clearlydescribed herein, have applications in the invention. Particularexamples of such proteins comprise TNF-α and CD40L, to name only a few.

-   23. The method of Claim 22, further comprising the step of washing    the dendritic cell before reintroducing the dendritic cell into the    animal.

Naturally, antigens having applications in the invention include:

-   -   a) a pathogen, or a fragment thereof,    -   b) a virus, or a fragment thereof; or    -   c) a tumor, or a fragment thereof.

Thus the instant invention readily permits one of ordinary skill in theart to treat an immune system related condition, which is associatedwith a particular antigen. In particular, the pulsing step of theinvention would including pulsing the dendritic cell with an antigenassociated with a particular immune system related condition. Thus whenthe dendritic cell is reintroduced into the animal, the animal's immuneresponse to the particular antigen is increased relative to the immuneresponse in an animal whose dendritic cells were not treated accordingto the teachings of the invention.

Accordingly, it is an object of the present invention to provide anisolated nucleic acid sequence which encodes a TRANCE protein or afragment thereof, and degenerate variants of such isolated nucleicacids. Disclosed herein are isolated nucleic acid sequences which encodemurine TRANCE and human TRANCE.

It is a further object of the present invention to provide an amino acidsequence for human and murine TRANCE, a conservative variant thereof ora fragment thereof, having utility in a pharmaceutical composition whichcan modulate immune response in a mammal, or procedure intended todiagnose an immune system related condition in a mammal.

It is a further object of the present invention is to provide anexpression vector which can be used to produce TRANCE, a conservativevariant thereof, a fragment thereof, or an analog or derivative thereofin a cell, wherein the expression vector comprises the isolated nucleicacid sequence of the present invention which encodes TRANCE, operativelyassociated with a promoter. A cell transfected with this vector can bemade to produce TRANCE, or a fragment thereof, which have applicationsas described above.

Yet another object of the present invention is to provide a fusionprotein containing a TRANCE, a conservative variant thereof, a fragmentthereof, or analog or derivative thereof, linked to a different protein,or a fragment of a different protein.

It is a further object of the present invention to provide antibodies tothe TRANCE and its subunits, and methods for their preparation,including recombinant means. Such antibodies include polyclonal,monoclonal and chimeric antibodies.

Yet still another object of the present invention is to provide amodulator of immune response in mammal, wherein this modulator regulatesthe life span of mature dendritic cells, and hence T cell activation. Inparticular, one object is to provide a TRANCE agonist modulator can bindto TRANCE-R on the surface of a mature dendritic cells and signal thecell, resulting in an upregulation of expression of Bcl-x_(L) in thecell. This protein inhibits apoptosis and hence prolongs the life spanof the mature dendritic cells of the mammal. As a result, the number ofT cells activated by a mature dendritic cell exposed to the TRANCEagonist modulator is greater than the number of T cells activated by amature dendritic cell not exposed to the TRANCE agonist modulator.Hence, immune response is increased.

Yet another object of the present invention is to provide a TRANCEantagonist modulator which can either bind to TRANCE on the surface of aT cell and prevent its binding to TRANCE-R on a mature dendritic cell,or binds to TRANCE mRNA, preventing its function, i.e., translation. Theantagonist TRANCE modulator can be an antibody having an TRANCE or afragment thereof as an immunogen. Moreover, an analog or derivative ofTRANCE, and a TRANCE fusion protein can also serve as an immunogen. Theantibody of the present invention which is a TRANCE antagonist modulatorcan be a polyclonal, monoclonal or chimeric antibody, the production ofwhich are all disclosed infra. A TRANCE antagonist modulator can also bean anti-sense nucleic acid molecule having at least phosphodiesteranalog bond which is complement to TRANCE mRNA such that the anti-sensemolecule of the present invention can bind to TRANCE mRNA and preventits function.

It is a still further object of the present invention to providepharmaceutical compositions for use in therapeutic methods wherein thepharmaceutical compositions comprise, or are derived from, TRANCEmodulators of the present invention, along with a pharmaceuticallyacceptable carrier. In particular, these pharmaceutical compositions ofthe present invention can be used to modulate the life span of maturedendritic cells in a mammal. Moreover, they can also be used to modulateT cell activation in a mammal, and hence immune response in a mammal.The present invention disclosed a pharmaceutical composition comprisinga TRANCE agonist modulator, as described above, and a pharmaceuticallyacceptable carrier thereof. Moreover, the present invention discloses apharmaceutical composition comprising a TRANCE antagonist modulator asdescribed above, and a pharmaceutically acceptable carrier thereof.

It is a still further object of the present invention to provide amethod to modulate immune response in a mammal, and in particular, tomodulate the life span of mature dendritic cells in the mammal, andhence modulate T cell activation in the mammal. In one such method, atherapeutically effective amount of the pharmaceutical compositioncomprising the TRANCE agonist modulator is administered to a mammal,along with an antigen. The antigen can be a virus, or a fragmentthereof, a pathogen or a fragment thereof, or a tumor, or a fragmentthereof. This method can be used to increase immune response againstsuch an antigen. Hence, conditions such as a virus like an adenovirus orHIV, cancer or an infection, can be treated with the method of theinvention.

Yet still another object of the present invention is to utilize thepharmaceutical composition invention containing a TRANCE antagonistmodulator to treat an immune system related condition, such asautoimmune disease. This method comprises administering to a mammal atherapeutically effective amount of the pharmaceutical compositionhaving the TRANCE antagonist modulator. As a result, the life span ofmature dendritic cells of the mammal, and hence its immune response, isdecreased. Such a method can also be used to treat a mammal sufferingfrom hypersensitivity to an allergen, whereby this method induces anergyin the mammal towards the allergen.

Another object of the present invention is to provide a method ofdiagnosing an immune system related condition in a mammal. This methodcomprises assaying a bodily sample of mammal to determine whether TRANCEis expressed therein. The bodily sample can be blood, or lymphoidtissue, such as lymph node; spleen or thymus tissue. The nucleic acidsand antibodies of the present inventions have utility in such an assay.

Also, another object is the use of the present invention in a genetherapy method to modulate the expression of TRANCE in the mammal. Themethod involves removing a hematopoietic stem cell from a mammal, anddestroying all remaining hematopoietic stem cells in the mammal. Thestem cell removed is transfected with the expression vector of thepresent invention, and then reintroduced into the mammal. As a result,the animal will ultimately be repopulated with T cells which expressTRANCE.

Still yet another object of the present invention is to increase immuneresponse in an animal against a particular antigen. An increase canresult via in vivo contact between a dendritic cell of the animalpreviously exposed to a particular antigen, and a modulator of immuneresponse, such as a TRANCE agonist described above. Also, immuneresponse against a particular antigen can be increased via a vivocontact between a modulator of immune response, such as a TRANCEagonist, and a dendritic cell previous exposed to the particular antigenex vivo.

Still yet another object of the invention is to utilize the heretoforeunknown cooperativity between TRANCE and a protein of the TNFsuperfamily to increase the viability of dendritic cells, and thusincrease an animals immune response towards a particular antigenrelative to a control animal whose dendritic cells were not pulsed withTRANCE and a member of the TNF superfamily, such as CD40L and TNF-α.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1. The cDNA nucleic acid molecule encoding human TRANCE (SEQ IDNO:1).

FIG. 2. The amino acid sequence of human TRANCE (SEQ ID NO:2).

FIG. 3. The cDNA nucleic acid molecule encoding murine TRANCE (SEQ IDNO:3).

FIG. 4. The amino acid sequence of murine TRANCE (SEQ ID NO:4).

FIG. 5. Identification of a gene defective in KIT50.1.9.1.

FIG. 5A. Differential screening of the 8-50.51 gene fragment and Nur77cDNA with probes from TCR-stimulated KMls8.3.5.1 (KMls8.3.5.1+) andTCR-stimulated KIT50.1.9.1 (KIT50.1.9.1+).

FIG. 5B. Northern analysis of the TRANCE transcript in control,unstimulated (−) and TCR-stimulated (+) KMls8.3.5.1 or KIT50.1.9.1 using8-50.51 cDNA as a probe. GAPDH was used as a control for poly A(+) RNAloading.

FIG. 6. Sequence analysis of the TRANCE gene.

FIG. 6A. The predicted amino acid sequence of the full length mouseTRANCE protein (mTRANCE) (SEQ ID NO:4) compared with the extracellulardomain of human TRANCE (hTRANCE) (SEQ ID NO:2). Dots indicate sharedidentity between the mouse and human protein and dashes indicate gapsbetween regions of homology. The transmembrane domain is underlined.Residues labeled with an asterisk (*) indicate a potential N-linkedglycosylation sites. The numbers in the left-hand column indicate theamino acid residue positions in the mTRANCE protein. Genbank accessionnumbers: mTRANCE, AF013170; hTRANCE (partial), AF013171.

FIG. 6B. Amino acid alignment of murine TRANCE (SEQ ID NO:4) with othermurine members of the TNF superfamily, such as mFasL (SEQ ID NO:5),mTRAIL (SEQ ID NO:6), mLT-Beta (SEQ ID NO:7), and mTNF-alpha (SEQ IDNO:8). Bars represent the β sheets as estimated from the TNF crystalstructure (31). Shaded residues are those that match the consensussequence. The numbers in the left-hand column indicate the residuepositions from the full length protein sequences. Dashes indicate gapsbetween regions of homology

FIG. 7. Expression and regulation of TRANCE

FIG. 7A. Left: Effect of FK506 and cycloheximide (CHX) on TCR-inducedupregulation of TRANCE and FasL by Northern analysis. T cell hybridomaswere stimulated on α-TCR Ab coated plates for the indicated amount oftime in the presence of media alone (−), FK506 (10 ng/mL) or CHX (1μg/mL). Right: Northern of TRANCE and FasL expression in LNTC stimulatedwith ConA+IL-2 or ConA+IL-2+α-CD3ε. The blots were stripped and reprobedwith GAPDH to normalize for the amount of loaded RNA.

FIG. 7B. Left: Northern analysis of TRANCE in various mouse tissues. 28Sand 16S ribosomal RNA is indicated. Equal amounts of RNA were loaded asdetermined with a 28S ribosomal RNA probe. Right: RT-PCR and Southernblot analysis of TRANCE in T and B cell enriched populations.Amplification of β-actin was used to control for the amount of RNAtemplate used in the PCR reaction.

FIG. 8. Characterization of the recombinant TRANCE-Ecto protein.

FIG. 8A. SDS-PAGE and Coomassie Brilliant Blue staining of purifiedTRANCE-Ecto. Molecular weight markers are indicated on the left of thefigure.

FIG. 8B. INK activation by TRANCE. Cells were purified as described inthe Materials and Methods and stimulated with 500 ng/mL of purifiedTRANCE-Ecto in 10% glycerol/PBS for the indicated amount of time on M2coated plates. As a negative control, thymocytes were treated with anequivalent volume of a 10% glycerol/PBS solution on M2 coated plates(control). JNK activation was assessed by incorporation of ³²P-ATP intopurified GST-c-Jun(1-79). The band intensities were quantified byphosphoimaging and presented as the fold induction over the unstimulatedsamples (0 min).

FIG. 9. TRANCE-R expression in various cell-types. Cells were preparedas described in the Materials and Methods section of Example II, infra.,and stained with 10 μg/mL of the hCD8-TRANCE recombinant protein (solidlines) or with secondary reagents alone (dotted line). Only viable cellsas determined by PI exclusion were gated and analyzed for TRANCE-Rexpression. Fresh DC were analyzed by two-color staining, after gatingon CD11c^(high) cells. Each staining was reproduced at least twice.

FIG. 10. TRANCE is a DC survival factor that upregulates

FIG. 10A. BMDC were cultured in complete medium in the presence orabsence of recombinant TRANCE (1 μg/mL) for 48 hours then visualizedunder an inverted light microscope.

FIG. 10B. Duplicate wells containing 3×10⁴ BMDC were cultured withincreasing doses of recombinant TRANCE in complete medium in flat-bottom96-well plates. The percentage of cell survival was assessed 48 h laterby trypan blue exclusion. The average of three experiments, and theSEMs, are shown.

FIG. 10C. 3×10⁴ BMDC were cultured in complete medium in the presence orabsence of recombinant TRANCE (1 μg/mL) or mCD8-CD40L ( 1/1000 of theculture supernatants). Cell viability was assessed daily by trypan blueexclusion. Representative data of three independent experiments areshown.

FIG. 10D 3×10⁴ GM-CSF and IL-4 stimulated human monocyte-derived DC werecultured for 2 days in monocyte conditioned medium (MCM) to generatemature DC (26). Thereafter, DCs were cultured in the presence or absenceof recombinant TRANCE (1 μg/mL) and cell viability was assessed each dayby trypan blue exclusion.

FIG. 10E 50 μg of protein extracted from BMDC cultured for 24 h asdescribed in FIG. 2C were analyzed for Bcl-2 and Bcl-x_(L) proteinexpression by western-blot analysis. Basal levels of Bcl-2 and Bcl-xLwere determined in day 8 BMDC (0 hr).

FIG. 11. Cell surface marker expression and T cell stimulatory functionof TRANCE treated BMDC.

FIG. 11A. 2.5×10³ BMDC were cultured with increasing doses of TRANCE ina final volume of 100 μl in triplicate in flat-bottom 96-well plates.After 48 hours 10⁵ purified allogeneic T cells in 100 μl were added ineach well and ³[H]-Thymidine incorporation was assessed after 3 days ofculture. One experiment of 3 are shown.

FIG. 11B. 2.5×10′ BMDC were cultured in the presence of or the absenceof TRANCE or CD40L for 48 h. After washing and counting the cells,dilutions of live cells were cultured with 10⁵ purified allogeneic Tcells and ³[H]-Thymidine incorporation was assessed after 3 days ofculture.

FIG. 11C. BMDC were cultured in complete medium for 24 h in the presence(solid lines) or absence (dotted lines) of soluble FLAG-TRANCE (1 μg/mL)and analyzed for the indicated surface markers expression by FACS aftergating the live cells. Similar results were obtained after 48 h ofculture.

FIG. 12. TRANCE does not induce the proliferation of B Cells. Triplicatewells of 2×10⁴ purified B cells were cultured in complete medium in thepresence of increasing doses of soluble TRANCE or CD40L in flat-bottom96 well plates. ³[H]-Thymidine incorporation was assessed after 2 daysof culture.

FIG. 13. TRANCE-R signaling is dependent on TRAF2. Thymocytes fromtransgenic mice expressing TRAF2.DN or control litter mates werestimulated with hCD8-TRANCE (1 μg/mL) on OKT8 (10 μg/mL) coated platesfor the indicated amount of time then assayed for JNK activity. Thedegree JNK activation was analyzed on a phosphorimager (Molecular ImagerSystem, Bio-Rad Laboratories, Hercules, Calif.) and plotted as foldinduction over time 0. Representative results of three independentexperiments are shown.

FIG. 14. Proliferative response of T cells to the antigen PPD taken fromthe Draining Lymph nodes, the Mesenteric Lymph nodes, and the Spleen.Immature BMDC were pulsed on day 6 for 6 hr with 10 μg/ml PPD and thenreplated in 100 mm dishes to induce maturation. On day 7, DC are furthercultured for 24 hr in the absence or the presence of TRANCE-hCD8 (1μg/ml) or CD40L ( 1/1000 viral sup). Cells were then extensively washed,counted, and resuspended in PBS. 200,000 cells in 50 μl PBS were theninjected in left hind footpad of syngeneic female mice. On day 6, 9 and14, draining LN (popliteal+inguinal), spleen and mesenteric LN cellswere restimulated in vitro with PPD and proliferation was assessed after3 days of culture. Each point represents the mean of 3 mice.

FIG. 15. TRANCE enhances recovering of migrating DCs in the lymph nodedraining site of injection. Unpulsed and TRANCE or CD40L-pulsed, mature,bone marrow derived DCs were labeled mice. Popliteal LNs were harvesteddaily and digested with collagenase. The LN cells were counted, stainedwith PE-conjugated CD11c, and analyzed on a FACScan. The absolute numberof injected DCs in each draining LN (CD11c+ve and FL-1 high) wascalculated. Each point represents the mean±SD of three mice in eachgroup and at each point. Similar results were obtained in threeexperiments.

FIG. 16. In vitro survival of mature DC. Mature bone-marrow or splenicDC were incubated in media with combinations of GM-CSF (10 ng/ml),mCD8-CD40L (1:100 dilution), hCD8-TRANCE (1 mg/mL), hIgG1 (10 mg/ml),TR-Fc (10 mg/ml), and mTNFα (50 ng/mL) for 72 h (bone marrow derived DC)or 24 h (spleen DC) and cell viability was measured by trypan blueexclusion. These concentrations of mCD8-CD40L, hCD8-TRANCE and mTNFαwere found to be saturating for survival of BMDC. Representative resultsof 3 independent experiments are shown.

FIG. 17. RT-PCR analysis of TRANCE mRNA expression in thymocytes andperipheral T-cell subsets. Lymph node T-cells (LNTC) were sorted by FACSbased on CD4, CD8 or CD44 expression. Naive and memory T cells wereidentified as CD44⁺ and CD44⁻ respectively. Thymocytes were sorted intosingle positive (SP, CD4⁺CD8⁻ and CD4⁻CD8⁺) and double positive (DP,CD4⁺CD8⁺) populations. The LNTC-sorted populations (1×10⁶) werestimulated on anti-CD3 mAb coated plates or were left unstimulated for3.5 h before their RNA was harvested. RT-PCR followed by southern blotanalysis with ³²P-labeled cDNA revealed the expression of TRANCE, CD40Land HPRT. HPRT levels normalized the amount of cDNA template used ineach PCR reaction.

FIG. 18. Specificity of hCD8-mTRANCE and TRANCE-R-Fc fusion molecules293-T cells transfected with vector alone (pcDNA), vector containingmTRANCE or the extracellular domain of TRANCE-R and stable cell lineswere cloned by limiting dilution. (A) Cells transfected with pFlag-CMV-1of pFlag-CMV-1/mTRANCE were incubated with 5 mg/ml of TRANCE.R-Fc (solidline) or normal hIgG1 (dotted line) followed by FITC-conjugatedanti-human Fc. (B) Cells transfected with pcDNA or pcDNA/TRANCE-R wereincubated with 10 mg/ml hCD8-mTRANCE (solid line) followed bybiotinylated anti-human CD8 and St-PE. Negative control cells wereincubated with the secondary Ab alone (dotted line). Cells were analyzedon a FACScan.

FIG. 19. Kinetics of TRANCE expression on CD4+ and CD8⁺ T-cellsactivated by anti-CD3 and anti-CD28. Purified lymph node T-cells werecultured in anti-CD3 coated (10 mg/ml) 96-well plates for the indicatedamount of time in the presence of 2.5 mg/ml of anti-CD28 mAb.Subsequently, cells were double stained with anti-CD4-PE or CD8-PE, andTR-Fc or control hIgG1 (5 mg/ml) followed by FITC-goat anti human IgGF(ab′)2, and binding was analyzed by FACS. Histograms reveal the bindingof TR-Fc (solid line) or hIgG1 (dotted line) on CD4⁻ and CD8⁺ gatedcells. One representative experiment of 4 is shown.

FIG. 20. The effect of CD28 mediated costimulation on TRANCE expressionon CD3-activated CD4+ and CD8⁻ T cells. Purified lymph node T cells werecultured as described in FIG. 19 in the presence or in the absence ofanti-CD28 mAb (2.5 mg/ml). TRANCE expression was assessed after 72 h ofculture. One representative experiment of 5 is shown.

FIG. 21. The expression of TRANCE by Th1 and Th2 clones. Resting oranti-CD3 activated (48 hours) Th1 and Th2 clones derived from DO11.10transgenic mice were stained with TR.R-Fc as described above.Representative results of two independent experiments are shown.

FIG. 22. IL-4 down regulates TRANCE expression on activated CD4− Tcells. Purified lymph node T cells were cultured as described above inthe presence or the absence of anti-CD28 mAb (2.5 mg/ml) and in thepresence or in the absence of murine rIL-4 (20 ng/ml). TRANCE expressionwas assessed after 72 h of culture. The results of one representativeexperiment of 4 are shown

FIG. 23 Activated T and B cell express low levels of TRANCE-R. (A)Purified lymph node T cells were cultured in anti-CD3 coated (10 mg/ml)96-well plate in the presence or in the absence of anti-CD28 mAb (2.5mg/ml), rIL-4 (20 ng/ml) and TGF-b1 (1 ng/ml). TRANCE-R expression wasassessed after 60 hours of culture using the hCD8-mTRANCE fusionmolecule as described in the Materials and Methods section. TRANCE-Rexpression was detected only after 48 h of simulation (B) Purifiedspleen B cells were cultured in 96-well plate in medium alone or in thepresence of soluble CD40L (1/100 dilution of insect cell supernatant) oranti-m chain Ab (0.5 mg/ml)+rIL-4 (20 ng/ml). After 48 h of culturecells were stained for CD40, CD95 (Fas), TRANCE-R and B7-2. Maximallevels of expression were detected between 24 and 60 h of stimulation.One representative experiment of 3 is shown

FIG. 24. TRANCE induces an array of cytokines in bone-marrow deriveddendritic cells (BMDC). (A) RNA was extracted from PBS and hCD8-TRANCE(2.5 mg/ml) treated BMDC and subjected to ribonuclease protection assaysas described in the Materials and Methods section to measure levels ofIL-1α, IL-1β, IL-1Ra IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-15, IL-1α,MIF, TNF-α, TNF-β (LT-α), LT-β, IFN-γ or IFN-β mRNA. Yeast tRNAcontrolled for non-specific probe protection. Representative results oftwo independent experiments are shown. (B) The GAPDH signal was used tocontrol for the amount of input RNA and to quantify the relativeexpression of cytokine mRNA by phosphorimaging.

DETAILED DESCRIPTION

As noted above, the present invention discloses an isolated nucleic acidmolecules, or degenerate variants thereof, which encode TRANCE. Examplesinclude an isolated nucleic acid molecule comprising a DNA sequence asset forth in FIG. 1 (SEQ ID NO:1), degenerate variants thereof,fragments thereof, or analogs or derivatives thereof. This moleculecorresponds to an isolated nucleic acid molecule which encodes humanTRANCE. Also disclosed herein is an isolated nucleic acid moleculehaving a DNA sequence as set forth in FIG. 3 (SEQ ID NO:3), degeneratevariants thereof, fragments thereof, or analogs or derivatives thereof,or, which corresponds to a isolated nucleic acid molecule which encodesmurine TRANCE

Moreover, nucleic acids hybridizable under standard hybridizationconditions to an isolated nucleic acid molecule comprising a DNAsequence as set forth in FIG. 1 (SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3),or degenerate variants thereof, are also included in the presentinvention.

Applicants have discovered that TRANCE is expressed on the surface of Tcells, and that mature dendritic cells of the immune system have asurface receptor, TRANCE-R. When the T cell interacts with a maturedendritic cell presenting an antigen, TRANCE interacts with TRANCE-R.This interaction causes an upregulation of the expression of Bcl-x_(L)in the mature dendritic cell, which inhibits apoptosis. As a result, thelife span of the mature dendritic cell is increased.

As used herein, the term “TRANCE” refers to an integral membrane boundprotein on the surface of T cells, which has been described [Wong et al.J. Bio. Chem 272:25190-25194 (1997); this publication is specificallyincorporated by reference in its entirety].

The term “TRANCE-R” refers to a TRANCE receptor located on the surfaceof mature dendritic cells.

An “agonist” of TRANCE according to the invention is a compound that (i)binds to TRANCE-R on the surface of mature dendritic cells; and (ii)activates signal transduction with the mature dendritic cell causing theupregulation of the expression of Bcl-x_(L) in the mature dendriticcell, which inhibits apoptosis in the cell. The activity of a TRANCEagonist can be evaluated in vitro by numerous methods, including, and byno means limited to, determining the life span of a mature dendriticcell exposed to the agonist and comparing it with the life span of amature dendritic cell not exposed to the agonist. A TRANCE agonist isexpected to increase the life span of mature dendritic cells, and thusbe useful in a treatment regimen for immune system related conditions,such as HIV or cancer.

An “antagonist” of TRANCE according to the invention is a compound thatblocks TRANCE (or a TRANCE agonist) signal transduction. In oneembodiment, the antagonist is an anti-TRANCE antibody which binds toTRANCE on the surface of T cells, and prevents the interaction of TRANCEwith TRANCE-R on the surface of mature dendritic cells. In anotherembodiment, the TRANCE antagonist is an anti-sense TRANCE nucleic acidcomprising at least one phosphodiester analog bond. A TRANCE antagonistis expected to prevent the upregulation of expression of Bcl-x_(L) inmature dendritic cells so that apoptosis is not inhibited in the cells.As a result, the life span of mature dendritic cells exposed to a TRANCEantagonist are decreased relative to the life span of mature dendriticcells which can interact with TRANCE. Moreover, this decrease in lifespan results in a decrease in T cell activation, and hence a decrease inimmune response.

Also, the term “pulsed” or “pulsing a cell” refer to permitting a cellin cell medium to make contact with a molecule in the cell medium, suchas a protein, for a predetermined period of time.

As used herein, the phrase “conservative variant” refers to a proteinhaving at least one amino acid residue of its sequence be mutated to aanother amino acid residue having chemical properties similar to theoriginal amino acid residue.

As used herein, the phrase “TNF-Superfamily” or TNF-family, or TNFcytokine family refer to a family of proteins that can regulateapoptosis in addition to an array of other biological effects, such ascell proliferation, and differentiation. Examples of proteins of theTNF-superfamily include, but are not limited to, TNF, LT-α, LT-β, FasL,CD40L, CD30L, CD27L, 4-1BBL, OX40L (1) and TRAIL/APO-2L. Members of thefamily exhibit the highest homology between their C-terminal, receptorbinding domains. The superfamily members are type II membrane proteinsthat act in an autocrine, paracrine or endocrine manner either asintegral membrane proteins or as proteolytically processed solubleeffectors.

Isolated TRANCE Protein and Fragments Thereof

Also included in the present invention are amino acid sequences ofTRANCE or fragments thereof. In one embodiment of the present invention,human TRANCE is a polypeptide comprising an amino acid sequence as setforth in FIG. 2 (SEQ ID NO:2), murine TRANCE is a polypeptide comprisingthe amino acid sequence of FIG. 4 (SEQ ID NO:4) or conservative variantsthereof. A polypeptide having an amino acid sequence as set forth inFIG. 2 (SEQ ID NO:2) or conservative variants thereof corresponds tohuman TRANCE.

Anti-TRANCE Antibodies

The present invention also includes antibodies having TRANCE, or afragment thereof, as an immunogen.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′), portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

Moreover, the antibodies to TRANCE included in the present invention aredetectably labeled in order permit their use in numerous diagnosticassays, such as immunoassays, which are explained herein. Suchdetectable labels include, but are not limited to, conjugation of theTRANCE antibody to alkaline phosphatase, peroxidase, or the integrationof radioactive isotopes into the structure of the antibody.

Expression of TRANCE in Expression Vectors of the Present Invention

According to the present invention, unicellular hosts can be transfectedwith an expression vector of the present invention. An expression vectorof the present invention can comprise an isolated nucleic acid moleculeencoding TRANCE, or degenerate variants thereof, operatively associatedwith a promoter. Such cells can be used to produce TRANCE for use in theelucidation of the molecular genetics of the TRANCE gene, and in thetreatment of immune system related conditions.

An isolated nucleic acid molecule comprising a DNA sequence as set forthin FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3), or degenerate variantsthereof, or an isolated nucleic acid molecule which is hybridizable toan isolated nucleic acid comprising a DNA sequence as set forth in FIG.1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) or degenerate variants thereof,under standard hybridization conditions, can also be used in anexpression vector of the present invention.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A DNA sequence is “operatively associated” to an expression controlsequence when the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively associated” includes having an appropriate start signal(e.g., ATG) in front of the DNA sequence to be expressed and maintainingthe correct reading frame to permit expression of the DNA sequence underthe control of the expression control sequence and production of thedesired product encoded by the DNA sequence. If a gene that one desiresto insert into a recombinant DNA molecule does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; preferablyat least about 20 nucleotides; and more preferably the length is atleast about 30 nucleotides.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell50:667).

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that do not share a commonevolutionary origin (see Reeck et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andnot a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refers to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A gene encoding TRANCE, whether genomic DNA or cDNA, can be isolatedfrom any source, particularly from a human cDNA or genomic library.Methods for obtaining these genes are well known in the art, asdescribed above (see, e.g., Sambrook et al., 1989, supra). Accordingly,any animal cell potentially can serve as the nucleic acid source for themolecular cloning of these genes. The DNA may be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), andpreferably is obtained from a cDNA library prepared from tissues withhigh level expression of the protein (e.g., a T cell cDNA library, sincethese are the cells that evidence highest levels of expression ofTRANCE), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired cell (See,for example, Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985,DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I,II). Clones derived from genomic DNA may contain regulatory and intronDNA regions in addition to coding regions; clones derived from cDNA willnot contain intron sequences. Whatever the source, the gene should bemolecularly cloned into a suitable vector for propagation of the gene.

Moreover, the promoter of the expression vector of the present inventioncan also includes immediate early promoters of hCMV, early promoters ofSV40, early promoters of adenovirus, early promoters of vaccinia, earlypromoters of polyoma, late promoters of SV40, late promoters ofadenovirus, late promoters of vaccinia, late promoters of polyoma, thelac the trp system, the TAC system, the TRC system, the major operatorand promoter regions of phage lambda, control regions of fd coatprotein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter,or promoters of yeast α mating factor.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding a TRANCE and/or its flanking regions.

Expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

TRANCE proteins of the invention, fragments thereof, conservativevariants thereof, or analogs, derivatives, or fusion proteins thereof,may be expressed chromosomally, after integration of the coding sequenceby recombination. In this regard, any of a number of amplificationsystems may be used to achieve high levels of stable gene expression(See Sambrook et al., 1989, supra).

A unicellular host into which the recombinant vector or vectorscomprising the nucleic acid encoding TRANCE is cultured in anappropriate cell culture medium under conditions that provide forexpression of the TRANCE by the cell. In one embodiment of the presentinvention, a nucleic acid encoding a TRANCE fusion gene is expressed ina baculovirus expression system as a fusion protein, wherein theextracellular domain of murine TRANCE (amino acid residues 245-316 ofFIG. 4 (SEQ ID NO:4)) was fused to human CD8α.

Any of the methods for the insertion of DNA fragments into a cloningvector may be used to construct expression vectors containing a geneconsisting of appropriate transcriptional/translational control signalsand the protein coding sequences. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombination(genetic recombination).

As is well known in the art, DNA sequences may be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

Expression of a TRANCE protein, fragment thereof, conservative variantthereof, or analog or derivative thereof of the invention may becontrolled by promoter/enhancer elements disclosed herein, but theseregulatory elements must be functional in the unicellular host selectedfor expression. Promoters which may be used to control gene expressioninclude, but are not limited to, the SV40 early promoter region (Benoistand Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter; and the animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift et al., 1984; Cell 38:639-646; Ornitz et al., 1986,Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987,Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFR/methotrexate co-amplification vector, suchas pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12 (1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV-LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible metallothioneinIIa gene promoter, hygromycin selectable marker; Invitrogen), pREP8(BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTRpromoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI,HindIII, NotI, XhoI, SfiI, and BamH1 cloning site, RSV-LTR promoter,G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter,hygromycin selectable marker, N-terminal peptide purifiable via ProBondresin and cleaved by enterokinase; Invitrogen). Selectable mammalianexpression vectors for use in the invention include pRc/CMV (HindIII,BstXI, NotI, Sbar, and ApaI cloning site, G418 selection; Invitrogen),pRc/RSV (HindIII, Spa, BstXI, NotI, XbaI cloning site, G418 selection;Invitrogen), and others. Vaccinia virus mammalian expression vectors(see, Kaufman, 1991, supra) for use according to the invention includebut are not limited to pSC11 (SmaI cloning site, TK- and β-galselection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI,SacII, KpnI, and HindIII cloning site; TK- and β-gal selection), andpTKgptF1S (EcoRI, PstI, SalI, AccI, HindIII, SbaI, BamHI, and Hpacloning site, TK or XPRT selection).

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Moreover,once a suitable host system and growth conditions are established,recombinant expression vectors of the present invention can bepropagated and prepared in quantity. As previously explained, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; and plasmid and cosmid DNA vectors, toname but a few.

In addition, a unicellular host cell strain may be chosen whichmodulates the expression of the inserted sequences, or modifies andprocesses the gene product in the specific fashion desired. Differenthost cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification (e.g.,glycosylation, cleavage [e.g., of signal sequence]) of proteins.Unicellular host cells of the present invention, include, but are notlimited to E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO,R1.1. B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.

Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, TRANCE activity. Furthermore, different vector/hostexpression systems may affect processing reactions, such as proteolyticcleavages, to a different extent.

Vectors are introduced into desired host cells by methods known in theart, e.g., transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J.Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent ApplicationNo. 2,012,311, filed Mar. 15, 1990).

A method for producing TRANCE a conservative variant thereof, a fragmentthereof, or analog or derivative thereof, wherein the method is includedin the present invention, comprises culturing a transfected ortransformed unicellular host described above under conditions thatprovide for expression of TRANCE, and recovering TRANCE from theunicellular host and the culture.

A Modulator of Immune Response

Also disclosed herein is a modulator of immune response in a mammal,wherein the modulator comprises a polypeptide having an amino acidsequence as set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof, or a fragment thereof, an analog orderivative of a polypeptide having an amino acid as set forth in FIG. 2(SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, or afragment thereof, a fusion protein wherein its amino acid sequencecomprises the amino acid sequence of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQID NO:4), conservative variants thereof or a fragment thereof, anantibody having an immunogen selected from the group consisting of apolypeptide having an amino acid sequence as set forth in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, or afragment thereof, an analog or derivative of a polypeptide having anamino acid as set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof, or a fragment thereof, a fusion proteinwherein its amino acid sequence comprises the amino acid sequence ofFIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservative variantsthereof or a fragment thereof, or an anti-sense TRANCE nucleic acidcomprising at least one phosphodiester analog bond.

There are two types of modulators disclosed herein. One type is anagonist of TRA-NCE, which increases the active life of mature dendriticcells relative to the life of mature dendritic cells not exposed to thismodulator, and hence increases T cell activation and overall immuneresponse in the mammal. This type of modulator is selected from thegroup consisting of a polypeptide having an amino acid sequence as setforth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservativevariants thereof, or a fragment thereof, an analog or derivative of apolypeptide having an amino acid as set forth in FIG. 2 (SEQ ID NO:2),FIG. 4 (SEQ ID NO:4), conservative variants thereof, or a fragmentthereof, a fusion protein wherein its amino acid sequence comprises theamino acid sequence of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof or a fragment thereof.

The other type of modulator is an antagonist of TRANCE. A TRANCEantagonist prevents the interaction of TRANCE with TRANCE-R and decreasethe active life of mature dendritic cells relative to the life of maturedendritic cells which can interact with TRANCE. Hence, a TRANCEantagonist modulator decreases T cell activation and overall immuneresponse in the mammal. This type of modulator comprises an anti-senseTRANCE nucleic acid comprising at least one phosphodiester analog bond,or an antibody having an immunogen selected from the group consisting ofa polypeptide having an amino acid sequence as set forth in FIG. 2 (SEQID NO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, or afragment thereof, an analog or derivative of a polypeptide having anamino acid as set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof, or a fragment thereof, a fusion proteinwherein its amino acid sequence comprises the amino acid sequence ofFIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID. NO:4), conservative variantsthereof or a fragment thereof.

The modulator of present invention also relates to genes encodinganalogs or derivatives of TRANCE, that have the same or homologousfunctional activity as the native TRANCE and homologs thereof from otherspecies. The production and use of derivatives and analogs orderivatives related to TRANCE are within the scope of the presentinvention. In one embodiment, the derivative or analog is functionallyactive, i.e., capable of exhibiting one or more functional activitiesassociated with a wild-type TRANCE of the invention. In anotherembodiment, the modulator is an analog or derivative of a TRANCE fusionprotein, which comprises protein domains from at least one specificprotein in a construct.

Conservative variants, analogs or derivatives of TRANCE can be made byaltering encoding nucleic acid sequences which encode TRANCE, bysubstitutions, additions or deletions that provide for functionallyequivalent molecules. Preferably, derivatives are made that haveenhanced or increased functional activity relative to that of nativeTRANCE.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence asgenomic or cDNA TRANCE can be used in the practice of the presentinvention. These include but are not limited to allelic genes,homologous genes from other species, and nucleotide sequences comprisingall or portions of the genes which are altered by the substitution ofdifferent codons that encode the same amino acid within the sequence,thus producing a silent change. Likewise, the TRANCE analogs orderivatives of the invention include, but, are not limited to, thosecontaining, as a primary amino acid sequence, all or part of the aminoacid sequence set forth in FIG. 2 (SEQ ID NO:2) or FIG. 4 (SEQ ID NO:4),including altered sequences in which functionally equivalent amino acidsare substituted for these sequences resulting in a conservative aminoacid substitution. For example, one or more amino acids within thesesequences can be substituted by another amino acid of a similarpolarity, which acts as a functional equivalent, resulting in a silentalteration. Substitutes for an amino acid within these sequences may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Amino acids containing aromatic ring structures arephenylalanine, tryptophan, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such alterations will not beexpected to effect apparent molecular weight as determined bypolyacrylamide gel electrophoresis, or isoelectric point.

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

The isolated nucleic acid sequence encoding TRANCE, or an analog orderivative thereof can be produced by various methods known in the art.The manipulations which result in their production can occur at the geneor protein level. For example, the cloned gene sequence can be modifiedby any of numerous strategies known in the art (Sambrook et al., 1989,supra). The sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog, care should be taken to ensurethat the modified gene remains within the same translational readingframe as the native gene, uninterrupted by translational stop signals,in the gene region where the desired activity is encoded.

Additionally, a nucleic acid sequence can be mutated in vitro or invivo, to create and/or destroy translation, initiation, and/ortermination sequences, or to create variations in coding regions and/orform new restriction endonuclease sites, or destroy preexisting ones, tofacilitate further in vitro modification. Preferably, such mutationsenhance the functional activity of the mutated gene product. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis (Hutchinson, C., etal., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986,Proc. Natl. Acad. Sci. U.S.A. 83:710), use of “TAB” linkers (Pharmacia),etc. PCR techniques are preferred for site directed mutagenesis (seeHiguchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology:Principles and Applications for DNA Amplification, H. Erlich, ed.,Stockton Press, Chapter 6, pp. 61-70).

Moreover, the present invention also includes derivatives or analogs ofTRANCE produced from a chemical modification. The TRANCE protein of thepresent invention may be derivatized by the attachment of one or morechemical moieties to the protein moiety. The chemically modifiedderivatives may be further formulated for intraarterial,intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal,pulmonary, topical or other routes of administration. Chemicalmodification of TRANCE may provide additional advantages under certaincircumstances, such as increasing the stability and circulation time ofthe chemically modified TRANCE and decreasing immunogenicity. See U.S.Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979. For a review,see Abuchowski et al., in Enzymes as Drugs (J. S. Holcerberg and J.Roberts, eds. pp. 367-383 (1981)). A review article describing proteinmodification and fusion proteins is Francis, 1992, Focus on GrowthFactors 3:4-10, Mediscript: Mountview Court, Friern Barnet Lane, LondonN20, OLD, UK.

Chemical Moieties For Derivatization. The chemical moieties suitable forderivatization may be selected from among water soluble polymers. Thepolymer selected should be water soluble so that the TRANCE analog orderivative does not precipitate in an aqueous environment, such as aphysiological environment. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable. One skilled in the art will be able to select the desiredpolymer based on such considerations as whether the polymer/componentconjugate will be used therapeutically, and if so, the desired dosage,circulation time, resistance to proteolysis, and other considerations.For TRANCE, these may be ascertained using the assays provided herein.

Examples of water soluble polymers having applications herein include,but are not limited to, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), dextran, poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols or polyvinyl alcohol.Polyethylene glycol propionaldenhyde may have advantages inmanufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached to TRANCE may vary, and oneskilled in the art will be able to ascertain the effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules TRANCE molecules willvary, as will their concentrations in the reaction mixture. In general,the optimum ratio (in terms of efficiency of reaction in that there isno excess unreacted component or components and polymer) will bedetermined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to TRANCE with consideration of effects on functional orantigenic domains of TRANCE. There are a number of attachment methodsavailable to those skilled in the art, e.g., EP 0 401 384 hereinincorporated by reference (coupling PEG to G-CSF), see also Malik etal., 1992, Exp. Hematol. 20:1028-1035 (reporting pegylation of GM-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group include lysine residues and theN-terminal amino acid residues; those having a free carboxyl groupinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulthydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecule(s). Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire N-terminally chemically modified TRANCE.Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to TRANCE molecules in the reaction mix,the type of pegylation reaction to be performed, and the method ofobtaining the selected N-terminally pegylated protein. The method ofobtaining the N-terminally pegylated preparation (i.e., separating thismoiety from other monopegylated moieties if necessary) may be bypurification of the N-terminally pegylated material from a population ofpegylated protein molecules. Selective N-terminal chemical modificationmay be accomplished by reductive alkylation which exploits differentialreactivity of different types of primary amino groups (lysine versus theN-terminal) available for derivatization in TRANCE. Under theappropriate reaction conditions, substantially selective derivatizationof TRANCE at the N-terminus with a carbonyl group containing polymer isachieved. For example, one may selectively N-terminally pegylate TRANCEby performing the reaction at a pH which allows one to take advantage ofthe pK, differences between the e-amino groups of the lysine residuesand that of the a-amino group of the N-terminal residue of TRANCE. Bysuch selective derivatization, attachment of a water soluble polymer toTRANCE is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of TRANCE and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to TRANCE. Polyethylene glycolproprionaldehyde, containing a single reactive aldehyde, may be used.

Also included in the modulator of the present invention is a fusionprotein wherein its amino acid sequence comprises an amino acid sequenceas set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservativevariants, or a fragment thereof. Fusion proteins are the product of anucleic acid created by the fusion of two distinct genes, or isolatednucleotide fragments of such genes. In one embodiment of the presentinvention, a soluble fusion TRANCE protein is disclosed which is aFLAG-tagged soluble form of TRANCE generated by cloning a PCR productencoding the TRANCE ectodomain (amino acid residues 72-316 of SEQ IDNO:4) into the HindIII-XhoI sites in the pFLAG/CMV-1 vector (Kodak). Inanother embodiment, a CD8-TRANCE fusion protein was produced, wherein athe extracellular domain of murine TRANCE (amino acid residues 245-316of FIG. 4 (SEQ ID NO:4)) was fused to human CD8α (amino acid residues1-82) and produced in a baculovirus expression system.

The modulator of the present invention also can be an anti-sense TRANCEnucleic acid comprising at least one phosphodiester analog bond. Ananti-sense molecule is comprised of RNA or DNA, or analogs orderivatives of RNA or DNA, and has a nucleotide sequence that iscomplementary to a specific RNA transcript of a gene. It is designed tohybridize to the specific RNA to form a duplex and block the function ofthe specific RNA, i.e., the translation of ANA. The anti-sense moleculeof the present invention can prevent the expression of TRANCE in Tcells. Hence, no interaction occurs between TRANCE and TRANCE-R on thesurface of mature dendritic cells. Signal transduction of TRANCE is thusblocked, and upregulation of the expression of Bcl-x_(L) in the maturedendritic cell is prevented. As a result, apoptosis is not inhibited,and the life span of mature dendritic cells is decreased relative to thelife span of mature dendritic cells wherein the TRANCE signaltransduction is not blocked.

Also included in the modulator of the present invention are antibodieshaving an immunogen comprising a polypeptide having an amino apolypeptide having an amino acid sequence as set forth in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, or afragment thereof, an analog or derivative of a polypeptide having anamino acid as set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof, or a fragment thereof, or a fusionprotein wherein its amino acid sequence comprises the amino acidsequence of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservativevariants thereof or a fragment thereof. Such antibodies include but arenot limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and an Fab expression library. The antibodies of theinvention may be cross reactive, e.g., they may recognize TRANCE fromdifferent species. Polyclonal antibodies have greater likelihood ofcross reactivity. Alternatively, an antibody of the invention may bespecific for a single form of TRANCE, such as human TRANCE having anamino acid sequence as set forth in FIG. 2 (SEQ ID NO:2), or murineTRANCE, having an amino acid sequence as set forth in FIG. 4 (SEQ IDNO:4).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to TRANCE polypeptide, fragment thereof,conservative variant thereof, or a derivative or analog thereof, orfusion proteins containing TRANCE or a fragment thereof. For theproduction of an antibody, various host animals can be immunized byinjection with the TRANCE polypeptide, or an analog or derivative (e.g.,fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the TRANCEpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the TRANCEpolypeptide, or fragment, analog or derivative thereof, or fusionprotein containing the amino acid residue sequence of TRANCE or afragment thereof, any technique that provides for the production ofantibody molecules by continuous cell lines in culture may be used.These include but are not limited to the hybridoma technique originallydeveloped by Kohler and Milstein [Nature 256:495-497 (1975)], as well asthe trioma technique, the human B-cell hybridoma technique [Kozbor etal., Immunology Today 4:72 1983); Cote et al., Proc. Natl. Acad. Sci.U.S.A. 80:2026-2030 (1983)], and the EBV-hybridoma technique to producehuman monoclonal antibodies [Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology [PCT/US90/02545]. In fact,according to the invention, techniques developed for the production of“chimeric antibodies” [Morrison et al., J. Bacteria 159:870 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)] by splicing the genes from a mouse antibody moleculespecific for a polypeptide together with genes from a human antibodymolecule of appropriate biological activity can be used; such antibodiesare within the scope of this invention. Such human or humanized chimericantibodies are preferred for use in therapy of human diseases ordisorders (described infra), since the human or humanized antibodies aremuch less likely than xenogenic antibodies to induce an immune response,in particular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. No. 4,946,778] can be adapted toproduce TRANCE polypeptide-specific single chain antibodies. Anadditional embodiment of the invention utilizes the techniques describedfor the construction of Fab expression libraries [Huse et al., Science246:1275-1281 (1989)] to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for a TRANCEpolypeptide, its derivatives, analogs or fusion proteins.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a TRANCE polypeptide, one may assay generatedhybridomas for a product which binds to a TRANCE polypeptide fragmentcontaining such epitope. For selection of an antibody specific to aTRANCE polypeptide from a particular species of animal, one can selecton the basis of positive binding with TRANCE polypeptide expressed by orisolated from T cells of that species of animal, and negative bindingwith TRANCE from other species.

The foregoing antibodies, particularly if they are detectably labeled,can be used in methods known in the art relating to the localization andactivity of the TRANCE polypeptide, e.g., for Western blotting, imagingTRANCE polypeptide in situ, measuring levels thereof in appropriatephysiological samples, etc., using any of the detection techniquesmentioned above or known in the art. Moreover, a detectable label for anantibody of the present invention includes, but is not limited to,conjugation of the antibody to an enzyme such as alkaline phosphatase orperoxidase, or the incorporation of radioactive isotopes into thestructure of an antibody of the present invention.

As explained above, a modulator of the present invention modulates theactive life of dendritic cells, and hence effects the immune responseand T cell activation in a mammal. It is important to note that thepresent invention includes modulators that are agonists of TRANCE thatincrease the life span of mature dendritic cells, and modulators thatare antagonists of TRANCE that decrease the life span of maturedendritic cells exposed to them relative to the life span of maturedendritic cells not exposed to a such a modulator.

In particular, modulators of the present invention which are agonists ofTRANCE and increase the life span of mature dendritic cells, increase Tcell activation, and hence increase immune response in a mammal, includea polypeptide having an amino acid sequence as set forth in FIG. 2 (SEQID NO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, or afragment thereof, an analog or derivative of a polypeptide having anamino acid as set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),conservative variants thereof, or a fragment thereof, a fusion proteinwherein its amino acid sequence comprises the amino acid sequence ofFIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservative variantsthereof or a fragment thereof.

Modulators of the present invention that are antagonists of TRANCEprevent the upregulation of expression of Bcl-x_(L) in mature dendriticcells so that apoptosis in those cells is not inhibited, resulting in adecrease of the life span of such mature dendritic cells, a decrease Tcell activation, and a decrease in immune response to an antigen. Onesuch TRANCE antagonist modulator of the present invention is ananti-sense TRANCE nucleic acid comprising at least one phosphodiesteranalog bond. Another TRANCE antagonist modulator of the presentinvention is an antibody having an immunogen comprising a polypeptidehaving an amino acid sequence of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ IDNO:4), conservative variants thereof, or a fragment thereof, an analogor derivative of a polypeptide having an amino acid as set forth in FIG.2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, ora fragment thereof, a fusion protein wherein its amino acid sequencecomprises the amino acid sequence of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQID NO:4), conservative variants thereof or a fragment thereof.

Pharmaceutical Compositions

Also provided in the present invention are pharmaceutical compositionscomprising modulators of the present invention, and a pharmaceuticallyacceptable carrier thereof. The phrase “pharmaceutically acceptable”refers to molecular entities and compositions that are physiologicallytolerable and do not typically produce an allergic or similar untowardreaction, such as gastric upset, dizziness and the like, whenadministered to a human.

Such pharmaceutical compositions may be for administration forinjection, or for oral, pulmonary, nasal or other forms ofadministration. In general, comprehended by the invention arepharmaceutical compositions comprising effective amounts of a modulatorof immune response of the invention with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions include diluents of various buffer content(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additivessuch as detergents and solubilizing agents (e.g., Tween 80, Polysorbate80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances(e.g., lactose, mannitol); incorporation of the material intoparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc. or into liposomes. Hylauronic acid may also beused. Such compositions may influence the physical state, stability,rate of in vivo release, and rate of in vivo clearance of the presentproteins and derivatives. See, e.g., Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712 which are herein incorporated by reference. The compositionsmay be prepared in liquid form, or may be in dried powder, such aslyophilized form.

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate apharmaceutical composition of the invention (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given by Marshall, K.In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter10, 1979, herein incorporated by reference. In general, the formulationwill include the modulator of the present invention and inertingredients which allow for protection against the stomach environment,and release of the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the modulator.The modulator may be chemically modified so that oral delivery of amodulator of the invention is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe modulator, where the moiety permits (a) inhibition of proteolysis;and (b) uptake into the blood stream from the stomach or intestine. Alsodesired is the increase in overall stability of the modulator andincrease in circulation time in the body. Examples of such moietiesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981,“Soluble Polymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-trioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the modulator, the location of release may be the stomach, the smallintestine (the duodenum, the jejunum, or the ileum), or the largeintestine. One skilled in the art has available formulations which willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine. Preferably, the release willavoid the deleterious effects of the stomach environment, either byprotection of the modulator or by release of the modulator in thepharmaceutical composition beyond the stomach environment, such as inthe intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The pharmaceutical composition can be included in the formulation asfine multi-particulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the pharmaceutical composition forcapsule administration could also be as a powder, lightly compressedplugs or even as tablets. The pharmaceutical composition could beprepared by compression.

Colorants and flavoring agents may all be included. For example, amodulator of the invention may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the pharmaceutical compositionwith an inert material. These diluents could include carbohydrates,especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may be also beused as fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the pharmaceuticalcomposition into a solid dosage form. Materials used as disintegratesinclude but are not limited to starch, including the commercialdisintegrant based on starch, “EXPLOTAB.” Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders may be used to hold the pharmaceutical composition together toform a hard tablet and include materials from natural products such asacacia, tragacanth, starch and gelatin. Others include methyl cellulose(MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of apharmaceutical composition of the invention to prevent sticking duringthe formulation process. Lubricants may be used as a layer between thepharmaceutical composition and the die wall, and these can include butare not limited to; stearic acid including its magnesium and calciumsalts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oilsand waxes. Soluble lubricants may also be used such as sodium laurylsulfate, magnesium lauryl sulfate, polyethylene glycol of variousmolecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the pharmaceuticalcomposition during formulation and to aid rearrangement duringcompression might be added. The glidants may include starch, talc,pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of a pharmaceutical composition of the invention intothe aqueous environment a surfactant might be added as a wetting agent.Surfactants may include anionic detergents such as sodium laurylsulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.Cationic detergents might be used and could include benzalkoniumchloride or benzethomium chloride. The list of potential non-ionicdetergents that could be included in the formulation as surfactants arelauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65and 80, sucrose fatty acid ester, methyl cellulose and carboxymethylcellulose. These surfactants could be present in the formulation of themodulator either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the modulator are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release oral formulation may be desirable. A pharmaceuticalcomposition of the invention can be incorporated into an inert matrixwhich permits release by either diffusion or leaching mechanisms, e.g.,gums. Slowly degenerating matrices may also be incorporated into theformulation. Some enteric coatings also have a delayed release effect.

Another form of a controlled release of a pharmaceutical composition ofthe invention is by a method based on the “OROS” therapeutic system(Alza Corp.), i.e. the pharmaceutical composition is enclosed in asemipermeable membrane which allows water to enter and push themodulator of the composition through a single small opening due toosmotic effects, thus delivering the modulator in vivo.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan-coater or in a fluidized bed orby compression coating.

Pulmonary Delivery. Also contemplated herein is pulmonary delivery of amodulator of the present invention. The pharmaceutical composition isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Other reports of thisinclude Adjei et al., 1990, Pharmaceutical Research, 7:565-569; Adjei etal., 1990, International Journal of Pharmaceutics, 63:135-144(leuprolide acetate); Braquet et al., 1989, Journal of CardiovascularPharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al., 1989,Annals of Internal Medicine, Vol. III, pp. 206-212 (al-antitrypsin);Smith et al., 1989, J. Clin. Invest. 84:1145-1146 (a-1-proteinase);Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings ofSymposium on Respiratory Drug Delivery II, Keystone, Colo., March,(recombinant human growth hormone); Debs et al., 1988, J. Immunol.140:3482-3488 (interferon-γ and tumor necrosis factor α) and Platz etal., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor). Amethod and composition for pulmonary delivery of drugs for systemiceffect is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 toWong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the “ULTRAVENT” nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the “ACORN II”nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the “VENTOLIN” metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the “SPINHALER” powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the modulator. Typically, each formulation is specific tothe type of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified modulator may also be prepared indifferent formulations depending on the type of chemical modification orthe type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the modulator of the presentinvention dissolved in water at a concentration of about 0.1 to 25 mg ofmodulator per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of themodulator caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the modulator suspended in apropellant with the aid of a surfactant. The propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the modulator and may also includea bulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation. The modulator should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung.

Nasal Delivery. Nasal delivery of a pharmaceutical composition of theinvention is also contemplated. Nasal delivery allows the passage of themodulator to the blood stream directly after administering thepharmaceutical composition to the nose, without the necessity fordeposition of the product in the lung. Formulations for nasal deliveryinclude those with dextran or cyclodextran.

As explained above, the present invention discloses a TRANCE agonistpharmaceutical composition comprising a modulator which is an agonist ofTRANCE comprising a polypeptide having an amino acid sequence as setforth in FIG. 2 (SEQ ID NO:2) or a fragment thereof, a polypeptidehaving an amino acid sequence as set forth in FIG. 4 (SEQ ID NO:4) or afragment thereof, the analog or derivative a polypeptide having an aminoacid as set forth in FIG. 4 (SEQ ID NO:4) or a fragment thereof, thefusion protein containing the amino acid sequence of FIG. 2 (SEQ IDNO:2) or a fragment thereof, and the fusion protein containing the aminoacid sequence of FIG. 4 (SEQ ID NO:4) or a fragment thereof, along witha pharmaceutically acceptable carrier thereof.

Moreover, the present invention discloses a TRANCE antagonistpharmaceutical composition comprising an antagonist of TRANCE comprisingan anti-sense TRANCE nucleic acid comprising at least one phosphodiesteranalog bond, or an antibody having an immunogen selected from the groupconsisting of the polypeptide having an amino acid sequence as set forthin FIG. 2 (SEQ ID NO:2) or a fragment thereof, the polypeptide having anamino acid sequence as set forth in FIG. 4 (SEQ ID NO:4) or a fragmentthereof, the analog or derivative of the polypeptide having an aminoacid sequence as set forth in FIG. 2 (SEQ ID NO:2) or a fragmentthereof, the analog or derivative of the polypeptide having an aminoacid sequence as set forth in FIG. 4 (SEQ ID NO:4) or a fragmentthereof, the fusion protein containing the amino acid sequence of FIG. 2(SEQ ID NO:2), or a fragment thereof, and the fusion protein containingthe amino acid sequence of FIG. 4 (SEQ ID NO:4), or a fragment thereof,and a pharmaceutically acceptable carrier thereof.

Methods for Treating Immune System Related Conditions with the PresentInvention

Pharmaceutical compositions of the present invention discussed above canbe used to treat immune system related conditions in a mammal. Inparticular, included in the present invention is a method to treat animmune system related condition in a mammal with a TRANCE agonistpharmaceutical composition, comprising the steps of exposing at leastone mature dendritic cell of the mammal to an antigen so that the atleast one mature dendritic cell can present the antigen on its surface,and administering to the mammal a therapeutically effective amount ofthe TRANCE agonist pharmaceutical composition. In this method, theantigen can include, but is not limited to, a pathogen or a fragmentthereof, a virus or a fragment thereof, or a tumor, or a fragmentthereof. Hence, the immune system related condition which can be treatedwith the present invention includes, but is not limited to viruses, suchas HIV or cancer.

As explained above, two costimulatory signals are necessary to activateT cells against a particular antigen presented on the surface of amature dendritic cell. One signal is from the antigen bound to an MHCmolecule on the surface of the mature dendritic cell. This complexinteracts with the T cell receptor complex on the surface of the T cell.The other signal results from molecules produced by the mature dendriticcell that bind to receptors on the T cell. The T cell becomes activatedupon receiving both signals, and undergoes an autocrine process whereinit separates from the dendritic cell and simultaneously secretes agrowth factor, such as IL-2, along with cell-surface receptors that bindto it. The binding of IL-2 to its receptor stimulates the T cell toproliferate, so long as it has already encountered its specific antigen.After receiving these signals, the activated T cell departs and theantigen presenting mature dendritic cell is available to activateanother T cell. Hence, TRANCE agonist modulators of the presentinvention mimic TRANCE, and induce upregulation of expression ofBcl-x_(L) in mature dendritic cells, which inhibits apoptosis of maturedendritic cells. As a result, the life span of the mature dendritic cellexposed to the TRANCE agonist modulator is increased relative to thelife span of mature dendritic cells which are not exposed to the TRANCEagonist modulator. This life span increase permits the mature dendriticcells presenting antigens to activate a greater number of T cells thanmature dendritic cells not exposed to TRANCE or a TRANCE agonistmodulator. Hence, the immune response of the mammal is ultimatelyincreased, since T cell activation contributes to the response.Consequently, this method of the present invention modulates T cellactivation in the mammal and permits the treatment of an immune systemrelated condition. The antigen may be a pathogen, or a fragment thereof,a virus, or a fragment thereof, or an antigen from a tumor.

The exposure of the mature dendritic cell to the antigen can occur invivo or in vitro. If it occurs in vivo, the antigen is administered tothe mammal so that mature dendritic cells of the mammal can interactwith the antigen and present it on their surfaces. If the exposureoccurs ex vivo, then at least one mature dendritic cell is removed fromthe mammal, exposed to the antigen ex vivo so that the antigen can bepresented on its surface, and then reintroduced into the mammal prior toadministering the TRANCE agonist pharmaceutical composition.

Moreover, the TRANCE antagonist pharmaceutical composition can also beused in a method to treat immune system related conditions. Inparticular, disclosed herein is a method for treating an immune systemrelated condition in a mammal comprising administering to the mammal atherapeutically effective amount of a TRANCE antagonist pharmaceuticalcomposition. In this embodiment, the immune system related condition isrelated to over-expression of TRANCE in the mammal. Autoimmune diseaseis an example of an immune system related condition involvingoverexpression of a protein receptor of the TNF superfamily. SinceTRANCE is a member of the TNF superfamily, its over expression can alsoresult in an immune system related condition. As a result, this methodcan be used to treat such a condition. Moreover, this method can be usedto treat hypersensitivity in a mammal towards an allergen. Since theTRANCE antagonist pharmaceutical composition decreases immune response,this method can be used to induce anergy in a mammal and decrease themammal's immune response towards an allergen.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in the S phaseactivity of a host cellular mass, or other feature of pathology such asfor example, elevated blood pressure, fever, or white cell count as mayattend its presence and activity.

Dosages. For all of the above pharmaceutical compositions, as furtherstudies are conducted, information will emerge regarding appropriatedosage levels for treatment of various conditions in various patients,and the ordinary skilled worker, considering the therapeutic context,age and general health of the recipient, will be able to ascertainproper dosing.

Gene Therapy

Also disclosed herein is a method for modifying levels of expression ofa TRANCE protein in a mammal, comprising the steps of removing at leastone hematopoietic stem cell from the mammal, destroying remaininghematopoietic stem cells in the mammal, transfecting the at least onehematopoietic stem cell with an expression vector comprising an isolatednucleic acid molecule which encodes a TRANCE protein such that thenucleic acid molecule becomes incorporated into the genome of thehematopoietic stem cell, forming a transfected hematopoietic stem cell,and introducing the transfected hematopoietic stem into the mammal sothat the transfected hematopoietic stem cell can self replicate anddifferentiate within the mammal. Such a method can be used to treat amammal in which TRANCE is not expressed.

The method for removing of a stem cell from a mammal is well known inthe art. Moreover, transfecting such a cell with an expression vector ofthe present invention such that a gene expressing TRANCE operativelyassociated with a promoter is incorporated into the genome of the stemcell is also included in the present invention. Destruction of remainingstem cells in the mammal can be done with numerous protocols, such asfor example, radiation treatments. It is important however, that themammal be kept in a germ free environment as its immune system will beunable to protect it from foreign viruses, bacteria and pathogens.

After all remaining stem cells of the mammal have been destroyed, thetransfected stem cell can be reintroduced into the mammal, wherein itreplicates and differentiates. As a result, the immune system isrepopulated with T cells that can express TRANCE.

In one embodiment of the present invention, this method is used tomodify TRANCE expression in a human, and the expression vector used totransfect the stem cell comprises the isolated nucleic acid sequence ofFIG. 1 (SEQ ID NO:1) operatively associated with a promoter.

Diagnostics

The antibodies or isolated nucleic acids of the invention can be used indiagnosis of diseases or disorders associated with apparent defects inTRANCE activity by evaluating the level of expression of a functionalTRANCE in a bodily sample. In particular, nucleic acid probes or PCRprimers can be used to verify expression of mRNA coding for TRANCE instandard protocols, such as Northern blots, Southern blots, and RT-PCRfollowed by a Southern blot. Moreover, anti-TRANCE antibodies of thepresent invention can be used in numerous immunoassays known in the artto detect the expression of TRANCE in a bodily sample. In particular,the antibodies of the present invention can be used to detect expressionof TRANCE in cells, and in some instances, to localize its expression onthe surface of T cells.

In specific embodiments, a deficiency in TRANCE activity may beevaluated in the context of a disease or disorder associated with adeficiency of a TNF protein. For example, a deficiency of the TNFsuperfamily protein CD40L results in an immune system related conditioncalled Hyper IGM syndrome. In another example Autoimmune HumanLymphoproliferative Syndrome (ALPS) is a condition caused by a mutationin the TNF superfamily protein Fas, such that the protein is notexpressed, or is expressed in a mutated form with either no activity ordecreased activity relative to the wildtype Fas protein. Consequently, adeficiency of TRANCE expression in an appropriate bodily sample of amammal is indicative of the presence of an immune system relatedcondition in the mammal. Appropriate bodily samples include, but are notlimited to, blood and lymphoid tissue such as lymph node tissue, spleentissue, or thymus tissue.

Screening a bodily sample as described above for expression of TRANCEcan be accomplished with antibodies of the present invention (includingantibodies which are TRANCE antagonist modulators) by techniques knownin the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbantassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, an anti-TRANCEantibody binding to TRANCE or a fragment thereof is detected bydetecting a label conjugated to the TRANCE antibody. In anotherembodiment, the TRANCE antibody binding is detected by detecting bindingof a secondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

Moreover, the isolated nucleic acid sequences of the present inventioncan be used to screen for the expression of TRANCE in a bodily sample.For example, The mRNA from a bodily sample can be isolated, andimmobilized on a solid support, such as nitrocellulose or polyvinylidenedifluoride (PVDF). This support can then be incubated with the isolatednucleic acid molecule of FIG. 1 (SEQ ID 1) or a fragment thereofconjugated to a label to form a “probe”. Numerous methods of labelingDNA are known in the art, including, but not limited to theincorporation of radioactive isotopes into the DNA. This probe iscomplementary to TRANCE mRNA in a sample and will form a duplex with it.Hence, the detection of this duplex on the solid support indicates thatthe TRANCE gene in the sample is being transcribed into mRNA, which, inturn, should be translated into a protein.

However, if no such duplex is detected, it is probable that the TRANCEis not being expressed, and hence the mammal lacks functional TRANCE.

Methods of Modulating Immune Response in an Antigen in an Animal

As explained above, Applicants have discovered that the interaction ofTRANCE with its receptor on the surface of mature dendritic cells causesthe cell's upregulation of expression of apoptosis suppressingBcl-x_(L). Hence, the cell's survivability upon exposure to TRANCE isincreased.

The present invention further extends to a method for modulating immuneresponse to a particular antigen in an animal, which exploits thisproperty of TRANCE. In particular, the method comprises the steps of:

-   -   a) pulsing immature dendritic cells from an animal with an        antigen ex vivo so that the immature dendritic cells present the        antigen on their surfaces;    -   b) inducing maturation of the dendritic cells ex vivo;    -   c) pulsing the mature dendritic cells with a modulator of immune        response ex vivo; and    -   d) introducing the mature dendritic cells into the animal.

In another embodiment, the present invention extends to a method forincreasing immune response to a particular antigen in an animal,comprising the steps of

-   -   a) removing the immature dendritic cell from the animal;    -   a) pulsing immature dendritic cells with TRANCE, a fragment        thereof, a conservative variant thereof, or analog or derivative        thereof;    -   b) pulsing the immature dendritic cell with the particular        antigen;    -   c) pulsing the immature dendritic cell with a protein which is a        member of the TNF-superfamily;    -   d) inducing the immature dendritic cell to mature; and    -   e) reintroducing the mature dendritic cell into the animal.

Contacting, or pulsing dendritic cells presenting the antigen on theirsurfaces with TRANCE or agonists thereof, increases the viability, e.g.,the life span, of the dendritic cells in vivo. As a result, the maturedendritic cells presenting the antigen on their surfaces interact with agreater number of T cells in vivo than they would have interacted withhad their survivability not been increased. Consequently, the immuneresponse in the animal towards the antigen presented on the surface ofthe dendritic cells is increased.

Any type of dendritic cell has applications in this method. Preferably,immature dendritic cells used in a method of the present invention arederived from bone marrow.

Moreover, the present invention extends to modulating immune responsetowards numerous types of antigens. Examples of antigens toward whichimmune response can be increased comprise pathogens, or fragmentsthereof, viruses or fragments thereof, or tumors, or fragments thereof.

Also, numerous methods of interacting the antigen with immaturedendritic cells ex vivo are encompassed by the present invention. Forexample, immature dendritic cells can be pulsed ex vivo with theantigen. In this method, the antigen is taken up by the cell, processed,and then presented on the cell's surface.

Another method of interacting immature dendritic cells with an antigenex vivo involves transfecting immature dendritic cells with anexpression vector comprising a nucleic acid which encodes the antigen,operatively associated with a promoter. In this method, expression ofthe expression vector within transfected ex vivo immature cells resultsin the production of the antigen, which is processed, and then presentedon the surface of the transfected immature dendritic cells.

Modulators of immune response described infra have applications in amethod of modulating immune response involving exposure to dendriticcells ex vivo. In particular, such modulators are TRANCE agonists, andcomprise:

-   -   a) a polypeptide having an amino acid sequence of FIG. 2 (SEQ ID        NO:2), FIG. 4 (SEQ ID NO:4), conservative variants thereof, or        fragments thereof;    -   b) an analog or derivative of a polypeptide having an amino acid        sequence set forth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID        NO:4), conservative variants thereof, or fragments thereof; or    -   c) a fusion protein having an amino acid sequence comprising an        amino acid sequence set forth in FIG. 2 (SEQ ID NO:2), FIG. 4        (SEQ ID NO:4), conservative variants thereof, or fragments        thereof.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

EXAMPLES Example 1 TRANCE is a Novel Ligand of the Tumor Necrosis FactorSuperfamily that Activates c-Jun N-Terminal Kinase in T Cells

A novel member of the tumor necrosis factor (TNF) superfamily,designated TRANCE, was cloned during a search for apoptosis-regulatorygenes using a somatic cell genetic approach in T cell hybridomas. TheTRANCE gene encodes a type II membrane protein of 316 amino acids with apredicted molecular weight of 35 kD. Its extracellular domain is mostclosely related to TRAIL, FasL and TNF. TRANCE is an immediate earlygene upregulated by TCR stimulation and is controlled bycalcineurin-regulated transcription factors. TRANCE is most highlyexpressed in thymus and lymph nodes but not in non-lymphoid tissues andis abundantly expressed in T cells but not in B cells.Cross-hybridization of the mouse cDNA to a human thymus library yieldedthe human homolog, which encodes a protein 83% identical to the mouseectodomain. Human TRANCE was mapped to chromosome 13q14 while mouseTrance was located to the portion of mouse chromosome 14 syntenic withhuman chromosome 13q14. A recombinant soluble form of TRANCE proteincomposed of the entire ectodomain induced c-Jun N-terminal kinase (JNK)activation in T cells but not in splenic B cells or inbone-marrow-derived dendritic cells. These results suggest a role forthis TNF-related ligand in the regulation of the T cell dependent immuneresponse.

The TNF superfamily currently includes TNF, LT-α, LT-β, FasL, CD40L,CD30L, CD27L, 4-1BBL, OX40L (1) and TRAIL/APO-2L (2, 3) which exhibitthe highest homology between their C-terminal, receptor binding domains.The family members are type II membrane proteins that act in anautocrine, paracrine or endocrine manner either as integral membraneproteins or as proteolytically processed soluble effectors. Binding totheir cognate receptors leads to the activation of several signaltransduction pathways: the cascade of caspase/ICE-like proteases, thenuclear factor-κB (NF-κB) family of transcription factors and themitogen-activated protein kinases including the c-Jun N-terminal proteinkinases (JNK), and the extracellularly-regulated kinases (ERK) (4-6).

The biochemical pathways activated by the TNF-related ligands arecoordinated to effect a diverse set of biological responses includingapoptosis, differentiation, proliferation, and survival (1). Caspasesexecute the biochemical events leading to apoptosis (4) whereas NF-κBappears to inhibit cell death (7). In addition to its anti-apoptoticrole, NF-κB regulates numerous genes, such as cytokines and adhesionmolecules, that are critical in triggering and maintainingimmune-mediated inflammatory responses (8). TNFR1, TNFR2, CD30, CD40,DR3/wsl-1/TRAMP/Apo-3, and the TRAIL receptor, when stimulated or overexpressed, recruit TRAF2, a signal transducing protein that activatesINK in vitro (9). Fas can activate JNK by recruiting the protein Daxx toits death domain (10). Thus, JNK activation appears to be a commonsignaling event downstream of TNF-related ligand/receptor binding. JNKis linked to lymphocyte activation and proliferation since it canactivate c-Jun, a component of the nuclear factor of activated T cells(NFAT) and activator protein-1 (AP-1) (11). Emerging evidence suggeststhat JNK is also critical in mediating apoptosis in non-lymphoid cellsin response to some (10μ, 12-14), but not all physiologic agonists,e.g., TNFR1 mediated cell death (9, 15).

The expression of TNF-related ligands on T cells are regulated bysignaling from the T cell receptor (TCR) and mediate many of itsbiological effects. FasL, TNF and CD30L are responsible for TCR-mediatedapoptosis of T cells and immature thymocytes (16, 17). Seven of the TNFsuperfamily members, in conjunction with TCR-stimulation, can enhance Tcell proliferation (1). Therefore, upregulation of TNF cytokine membersand their receptors by the TCR may provide an autocrine costimulatorymechanism to enhance the cells' own proliferation after stimulation withantigen (1). The TCR also upregulates TNF-related ligands for thepurposes of B cell co-stimulation, protection against Igantigen-receptor induced apoptosis and antibody isotype switching(18-20), dendritic cell activation and differentiation (21), and ofinducing apoptosis in virally infected cells or transformed cells (22).

To investigate the molecular regulation of TCR-mediated apoptosis acloning strategy based on somatic cell genetics (23) was used, in whichgene expression in mutant T cell hybridomas, resistant to TCR-mediatedcell death yet capable of other receptor-associated functions (e.g. IL-2secretion), is compared with gene expression in wild-type cellssensitive to TCR-mediated cell death. Such a strategy should yield genesassociated with apoptosis and not activation although it is possible toobtain genes involved in other processes. This technique was usedsuccessfully to clone the gene TDAG51, a gene required for Fasexpression and TCR-mediated cell death (24). Using similar methods newmember of the TNF superfamily, designated TRANCE (TNF-relatedactivation-induced cytokine) was cloned, which is predominantlyexpressed on T cells and in lymphoid organs and is controlled by the TCRthrough a calcineurin-regulated pathway. A soluble form of the ligandconsisting only of the extracellular domain can activate c-JunN-terminal kinase (INK) specifically in T cells but not in B cells orbone-marrow derived dendritic cells. These results suggest that TRANCEplays a specific role in regulating T cell functions.

Materials and Methods

Subtractive hybridization and differential screening. 1×10⁸ KMls8.3.5.1or KIT50.1.9.1 T cell hybridomas, were incubated on 15 cm plates coatedwith 5 μg/mL of H57-597 (α-TCR Ab) as previously described (24). PolyA(+) RNA was extracted using a FastTrack 2.0 mRNA isolation kit(Invitrogen) and 2 μg from TCR-stimulated KMls8.3.5.1 (KMls8.3.5.1+) andTCR-stimulated KIT50.1.9.1 (KIT50.1.9.1+) was used to make tester anddriver cDNA, respectively. Suppression subtractive hybridization wasperformed using the PCR-select cDNA subtraction kit according to themanufacturer's protocol (Clontech). Briefly, tester and driver wasdigested with RsaI and the tester was ligated to adapter DNA. After twohybridizations with the tester and driver cDNA (20 h and 8 h) theresulting mixture was diluted 1:1000 and amplified by PCR using flankingand nested primers to produce a subtracted and normalized PCR fragmentlibrary. The efficiency of subtraction was verified via Southern blotanalysis of the unsubtracted and subtracted PCR products using a³²P-labeled GAPDH cDNA probe. 26 primary cycles and 18 secondary cyclesof PCR amplification yielded the greatest signal:noise ratio estimatedby comparing the amount of PCR product synthesized to the amount ofGAPDH in the subtracted product. Using these conditions, subtracted PCRproducts were TA cloned into the pCR2.1 plasmid (Invitrogen). To screendifferentially expressed products 100 ng of plasmid DNA containing thesubtracted fragments were immobilized on duplicate nitrocellulosefilters using a slot blot apparatus (Schleicher and Schuell) andhybridized to cDNA probes (1×10⁷ cpm/mL) derived from eitherKIT50.1.9.1+ or KMls8.3.5.1+ poly A(+) RNA. Signals were quantifiedusing a phosphorimager (Molecular Dynamics).

Full length cloning of murine and human TRANCE cDNA. A subtracted cDNAfragment, designated 8-50.51, which scored positive in the differentialscreening assay was used to screen a λZAP cDNA library derived fromKMls8.3.5.1+ (24). The longest clone (2.2 kb) was sequenced with aSequenase 2.0 kit (United States Biochemical) over both sense andanti-sense directions using a series of oligonucleotide primers. Toclone the human homolog a BamHI-BamHI fragment corresponding to TRANCE(nt 366-1035) was used to screen 1×10⁶ phage from a λgt11 5′-StretchPlus human leukemia library (Clontech) using low stringencyhybridization conditions. A partial human clone was sequenced using thesame method described for murine TRANCE.

Mouse cell purification. All cells were harvested from 4-8 week oldBALB/c mice (The Jackson Laboratory). T cell enrichment: T cells werepurified from 5×10⁷ lymph node cells using a T cell enrichment kit(Biotex). B cell enrichment: 5×10⁷ splenocytes were negatively selectedfor T cells via magnetic beads conjugated to anti-mouse Thy 1.2following the manufacturer's protocol (Dynabeads Thy 1.2, Dynal).Bone-marrow derived-dendritic cell enrichment (BMDC): Mature BMDC wereisolated as previously described (25). Proliferating and apoptotic lymphnode T cells (LNTC): LNTC were harvested and treated with concanavalin A(ConA; 5 μg/mL) plus IL-2 (10 U/mL) for 48 h and then with IL-2 alone(50 U/mL) for 48 h to yield proliferating T cells (17). To induce celldeath, the proliferating T cells were incubated on α-CD3ε (145-2C11)coated plates for 6-72 hours as previously described (17). Using theseconditions, ˜50% of the cells are dead by 48 h versus ˜5% cell death inthe cells treated with ConA plus IL-2 alone. The purity of T, B and BMDCenriched fractions was tested by FACS and in all cases was greater than90%.

Northern Analysis and Semi-Quantitative PCR. Expression and regulationof TRANCE in T cell hybridomas was determined by Northern blot analysisof poly A(+) RNA extracted at the indicated time points from thefollowing samples: unstimulated or TCR-stimulated cells either in thepresence of media alone, FK506 (10 ng/mL; Fujisawa USA) or cycloheximide(1 μg/mL; Sigma). The 8-50.51 fragment was used as a probe. To determineTRANCE expression in mouse tissues or in stimulated LNTC, total RNA wasextracted from various organs or cells as previously described (24) and20 μg from each sample was analyzed by Northern blot using the TRANCEfull length cDNA as a probe. A 28S ribosomal RNA probe or a GAPDH cDNAprobe was used to control for RNA loading. For semi-quantitative PCRanalysis total RNA was extracted from T or B cell enriched fractionsusing the RNA Isolation Kit (Stratagene) and first strand cDNA wastranscribed from 1 μg of RNA using Superscript RT (Gibco BRL) followingthe protocol provided by the supplier. The first strand reaction wasdiluted 1:100, allowing amplification to occur as linear function ofstarting concentrations, and was subjected to PCR using the followingconditions: β-Actin: (sense: 5′-ATG AAG ATC C.G. ACC GAG CG-3″ (SEQ IDNO:9), antisense: 5′-TAC TTG CGC TGA GGA GGA GC-3′ (SEQ ID NO:10), 94°C. 30 sec, 50° C. 1 min, 72° C. 1 min for 30 cycles). TRANCE: sense:5′-CCT GAG ACT CCA TGA AAA CGC-3′ (SEQ ID NO:11), antisense: 5′-TAA CCCTTA GTT TTC CGT TGC-3′ (SEQ ID NO:12), 94° C. 30 sec, 52° C. 1 min, 72°C. 1 min for 30 cycles). The PCR products were analyzed by Southern blotas previously described (24).

Expression and Purification of Soluble TRANCE. A FLAG-tagged solubleform of TRANCE was generated by cloning a PCR product encoding theTRANCE ectodomain (amino acid residues 72-316 of FIG. 2 (SEQ ID NO:2))into the HindIII-XhoI sites in the pFLAG/CMV-1 vector (Kodak). The openreading frame and FLAG fusion was confirmed by sequencing. 293T cellswere transfected with the expression construct (20 μg/10 cm plate) bythe calcium phosphate method. Supernatant was harvested 72 h later,passed through a 0.45 μm filter, incubated with the α-FLAG M2 affinitygel (Kodak) and eluted with the FLAG peptide (250 μg/mL; Kodak) asoutlined in the manufacturer's protocol. The eluant was dialyzed againstPBS, adjusted to 10% glycerol and the protein concentration wasascertained in a BCA protein assay (Pierce).

Chromosomal localization of murine and human TRANCE. Human TRANCEmapping: A Genebridge 4 radiation hybrid mapping panel was obtained fromResearch Genetics, Inc. (Huntsville, Ala.). Hybrid DNA was subjected toPCR (94° C. 20 sec, 55° C. 15 sec, 72° C. 1 min, for 30 cycles) withprimers derived from the 3′-UTR of the human TRANCE mRNA. Analysis ofthe data was performed using the radiation hybrid mapping server at theWhitehead Institute/MIT Center for Genome Research as previouslydescribed (26). Murine TRANCE mapping: Murine TRANCE was mapped using anintersubspecific backcross. A TRANCE specific genomic DNA fragment of582 bp was amplified by PCR using synthetic oligonucleotide primers(5′-ACC CAG ATG GAC TTC TGT GG-3′ (SEQ ID NO:13), 5′-TTT CCT TCG ACG TGCTAX CG-3′ (SEQ ID NO:14), and a single stranded conformationpolymorphism between 57BL/6J and CAST/Ei mice was detected in MDE gelsas previously described (27). The polymorphism was mapped on a panel ofDNA from 57 C57BL/6J×CAST/Ei)F1×57BL/6J backcrossed mice, donated by TheJackson Laboratory Mouse Mutant Resource, which contains a large numberof previously typed markers on all chromosomes (28).

c-Jun N-terminal kinase assays. 2-5×10⁶ cells were incubated for 1-2hours at 37° C., 5% CO₂ on plates coated with the α-FLAG M2 antibody (10μg/mL). The cells were treated with either soluble TRANCE in 10%glycerol/PBS solution or an equal volume of 10% glycerol/PBS solutionbefore harvesting at the indicated time points and frozen in a dryice/ethanol bath. Cells were lysed with Triton Lysis Buffer [20 mMTris.Cl (pH 7.5), 137 mM NaCl, 1 mM PMSF, 5 mM EDTA, 2 mM EGTA, 1 mMNa₃VO₄, 25 mM β-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate,15% glycerol, 1% Triton X-100], spun down in a microcentrifuge to removecell debris, and supernatants were incubated with goat α-JNK1 Ab (0.3μg; Santa Cruz Biotechnology) for 2 h at 4° C. Protein A sepharose wasadded for 1 h and the beads were washed 2 times with Triton Lysis Bufferthen 2 times with JNK Reaction Buffer [25 mM HEPES (pH 7.4), 25 mMβ-glycerophosphate, 25 mM MgCl₂, 2 mM DTT, 0.1 mM Na₃VO₄]. For thekinase reaction, 30 μL of JNK Reaction Buffer containing 1.5-3.0 μg ofpurified GST-c-Jun(1-79) (generously donated by Dr. H. Hanafusa, TheRockefeller University), 0.5 μCi of γ-³²P ATP and ATP (20 μM) wasincubated with the immunoprecipitated JNK for 20 min at 30° C. Thereactions were stopped with 2× loading buffer, boiled for 5 min and runon a 12% SDS-PAGE gel as previously described. (29)

Results and Discussion

Identification of mouse and human TRANCE. The molecular defects inKIT50.1.9.1, a mutant T cell hybridoma resistant to TCR-mediatedapoptosis (23) were investigated by comparing its gene expression withthat of KMls8.3.5.1, the parental cell line sensitive to TCR-mediatedapoptosis. Differentially expressed genes between TCR-stimulatedKIT50.1.9.1 (KIT50.1.9.1+) and TCR-stimulated KMls8.3.5.1 (KMls8.3.5.1+)were isolated using suppression subtractive hybridization, a cDNAsubtraction technique based on suppression PCR that is sensitive to raretranscripts (30). After subtracting KIT50.1.9.1+ cDNA from KMls8.3.5.1+cDNA a mini-library was generated by randomly subcloning the subtractedPCR products. The plasmid library was then subjected to differentialscreening using KIT50.1.9.1+ cDNA and KMls8.3.5.1+ cDNA as probes. Ofthe 347 plasmids screened, 76 produced a stronger signal with theKMls8.3.5.1+ probe than with the KIT50.1.9.1+ probe. One positive,designated 8-50.51, is shown in FIG. 5A. In contrast, Nur77, a genewhose expression is induced normally in both cells produced similarsignals with both probes indicating that an equivalent amount of labeledprobe was used from each cell line. Sequencing of 8-50.51 revealed an 87bp DNA fragment with no homology to any genes in the Genebank database.To confirm differential expression of 8-50.51, a Northern blotcontaining unstimulated and TCR-stimulated KMls8.3.5.1 and KIT50.1.9.1poly A(+) RNA was probed. The probe identified a 2.2-2.3 kB message thatwas highly induced in TCR-stimulated in KMls8.3.5.1 but only weaklyinduced in TCR-stimulated KIT50.1.9.1 (FIG. 5B).

Using 8-50.51 as a probe we screened a KMls8.3.5.1+ cDNA library wasscreened to obtain a full length clone. The full length cDNA (FIG. 6A)is 2237 bp in length with a canonical Kozak consensus sequence startingat 137 bp from the 5′ end of the clone. This translation initiation sitepermits the synthesis of a 316 amino acid protein with a hydrophobictransmembrane domain and no identifiable signal sequence stronglysuggesting a type II integral membrane protein topology. A comparison ofextracellular domains revealed similarity of the protein with mouseTRAIL (20%), FasL (19%) and TNF (17%). Alignment with selected membersof the TNF superfamily demonstrates high identity, especially in regionsforming the β strands as estimated from the TNF crystal structure (31)(FIG. 6B). Due to the clear similarity of this gene with the TNFsuperfamily members, this protein was termed TRANCE. A FLAG-tagged fulllength protein with an estimated molecular weight of 35 kD was detectedby Western blotting as a ˜45 kD band suggesting that TRANCE ispost-translationally modified. Putative N-linked glycosylation sites areindicated (FIG. 6A). The FLAG-tagged TRANCE could not beimmunoprecipitated with Fas, DR3/wsl-1/TRAMP/Apo-3, CD30, TNFR2, orHVEM/ATAR immunoadhesins suggesting that TRANCE does not bind to thesereceptors. A partial human TRANCE cDNA, cloned from a human thymus cDNAlibrary, is 83% identical to the mouse TRANCE ectodomain suggesting thatthe function of this gene is highly conservative between mouse andhuman.

Regulation and tissue distribution of TRANCE. The signaling capabilitiesand biological functions of the TNF superfamily appear redundant. Yetspecificity clearly exists, as shown by gene-knockout studies, in whichthe deletion of one superfamily member cannot be fully compensated bythe others. Specificity may be achieved by restricting the expression ofthese genes to particular cells and tissues and or by linking theirinduction to different regulatory pathways. Temporal regulation of theTNF superfamily members may also be important in properly coordinatingtheir biological effects in vivo (1). The regulation of TRANCE inductionby the TCR was studied in T cell hybridomas with cycloheximide (CHX), aninhibitor of translation, and FK506, a FKBP ligand that inhibitscalcineurin (PP-2B) (FIG. 7A, Left). Without inhibitors, TRANCEexpression began 1 h after TCR-stimulation and reached a maximal levelat 2.5 h. FasL was also highly induced by TCR-stimulation, however, itsexpression began at a later time point. CHX failed to inhibit theinduction of TRANCE by the TCR indicating that TRANCE is an immediateearly gene. In contrast, CHX completely abrogated the induction of FasLby the TCR. Thus, TRANCE, like TNF (32), is an immediate early gene witha relatively rapid onset of expression after TCR stimulation whereasFasL induction is delayed and requires de novo protein expression forits synthesis. Cyclosporin A, and FK506, both inhibitors of calcineurin,repress TCR-mediated TNF induction (32) and NFATp deficient mice fail toupregulate FasL, CD40L and TNF expression in response to TCR stimulation(33). Therefore, FasL, CD40L and TNF appear to be regulated bycalcineurin-dependent signaling pathways involving the NFAT family oftranscription factors. In the presence of FK506, the induction of TRANCEand FasL is blocked (FIG. 7A, Left) suggesting that TRANCE, like severalother TNF-related ligands, is controlled by NFAT proteins.

To examine TRANCE regulation in non-transformed cells, Northern analysiswas performed on concanavalin A (ConA) and IL-2 stimulated LNTC, a modelof antigen-mediated T cell proliferation and on proliferating LNTCre-stimulated with α-CD3E Ab, a model of peripheral T cell clonaldeletion (17). ConA and IL-2 stimulated T cells express relatively lowamounts of TRANCE message whereas FasL expression is high (FIG. 7A,Right). However, after re-stimulation with an α-CD3ε Ab TRANCE wassignificantly upregulated suggesting that TRANCE may play a role inantigen-induced T cell death.

Northern blot analysis revealed that TRANCE expression is restricted tothe thymus and lymph node (FIG. 7B, Left). This pattern of expressiondiffers from the pattern exhibited by TRAIL/Apo-2L and FasL, which areexpressed in both lymphoid and non-lymphoid organs, but is similar tothe pattern exhibited by lymphotoxin-β, which is restricted to spleen.In addition, TRANCE is abundant in lymph node-derived T cells (LNTC) butnot in splenic B cells (FIG. 7B, Right). Thus, TRANCE is expressedspecifically in T cells and in T cell rich organs, although itsexpression in other cell-types cannot be ruled out.

Chromosomal Mapping of TRANCE. The murine TRANCE locus was mapped tomouse chromosome 14 by use of an intersubspecific backcross (27, 28). In57 backcross mice TRANCE showed two recombinants with the Rb1 locus (Lod13.4) and nine recombinants with Rps10-rs4 (Lod>>6.4). After inferringmarker genotypes from recombinant mice, incorporating other markers andminimizing double crossovers, the gene order and map distances (cM±SE)were:Rb1-(1.5±1.0)-TRANCE-(1.5±1.1)-Rps10-rs4-(3.7±1.6)-Rpl36-rs2-(6.4±2.1)-Rpl7-rs2-(4.2±1.7)-Dct.TRANCE is located on mouse chromosome 14 near a non-MHC locussuggestively linked to autoimmune nephritis in NZB mice (34),implicating TRANCE in the regulation of immune-tolerance. Human TRANCEwas localized by radiation hybrid mapping at 3.98 cR (approximately 800kB) from the marker, CHLC.GATA6B07 (D13S325), located at 117 cR on theWI radiation hybrid framework of chromosome 13. Superposition of thismap with the cytogenetic map of human chromosome 13 allowed theassignment of TRANCE to chromosomal band 13q14.

Biochemical function of TRANCE. A soluble fusion protein comprising theentire ectodomain of TRANCE fused to an N-terminal FLAG epitope(TRANCE-Ecto) was constructed to examine the biochemical function ofTRANCE and to identify the cellular targets that respond to thisprotein. TRANCE-Ecto, when expressed in 293T cells and purified tohomogeneity, has an apparent molecular weight of ˜37 kD by SDS-PAGEanalysis (FIG. 8A). Since its calculated molecular weight is 27.5 kD,this data suggests that the TRANCE-Ecto protein is post-translationallymodified similarly to the FLAG-tagged membrane bound protein. JNK is asignal transducing molecule commonly activated by TNF-related ligands.Therefore, JNK activation was assessed by the soluble TRANCE protein inthymocytes, LNTC, purified splenic B cells and bone-marrow-deriveddendritic cells (BMDC). JNK is rapidly activated in thymocytes (3-foldinduction at 10 min), LNTC (2-fold induction at 5 min) (FIG. 4B) and Tcell hybridomas (2-fold at 10 min). In contrast, no effect was observedin B cells, or in BMDC (FIG. 4B). B cells and BMDC may not be sensitiveto soluble TRANCE at the concentration used in this assay due to thelack of an adequate number of cell surface receptors. Anotherpossibility is that only certain cell types express JNK-activatingsignal transducing molecules. These results suggest that the TRANCErecombinant protein is biologically active and appears to stimulate JNKspecifically in cells of the T cell lineage.

Described herein is the cloning of a novel member of the TNF cytokinegene family whose expression is restricted to T cells and lymphoidorgans and can participate in signaling to T cells implicating TRANCE inthe regulation of T cell dependent immune responses. TRANCE was obtainedthrough a genetic screen and it appears associated with cell death andnot cell survival or proliferation (FIG. 5B and FIG. 7A, Right). Due tothe multi-functional role other TNF-related molecules exhibit it islikely that TRANCE plays a role in cell activation, proliferation,survival or death depending on the context in which it is expressed andthe nature of the target cell. In support of this, TRANCE activates JNK,a kinase with pleiotropic biological effects.

Example II TRANCE, a New TNF SuperFamily Member Predominantly Expressedin T Cells, is a Dendritic Cell Specific Survival Factor

TRANCE is a new member of the TNF family that is induced upon T cellreceptor engagement and activates c-Jun N-terminal kinase (JNK)following interaction with its putative receptor (TRANCE-R) (Wong, etal., J. Biol. Chem., 272; 25190-25194). TRANCE expression is restrictedto lymphoid organs and to T cells. Disclosed herein is a showing thathigh levels of TRANCE-R are detected on mature dendritic cells (DC) butnot on freshly isolated B cells, T cells or macrophages. Signaling byTRANCE-R appears dependent on TNF receptor-associated factor 2 (TRAF2),since INK induction is impaired in cells from transgenic mice overexpressing a dominant negative TRAF2 protein. TRANCE inhibits apoptosisof mouse bone-marrow derived DC and human monocyte-derived DC in vitro.The resulting increase in DC survival is accompanied by a proportionalincrease in DC-mediated T cell proliferation in an MLR. TRANCEupregulates Bcl-x_(L) expression thus enhancing DC survival. TRANCE doesnot induce the proliferation of B cells or increase the survival of T orB cells. Therefore, TRANCE is a new DC restricted survival factor thatmediates T cell-DC communication and provides a tool to modulate DCactivity, and hence immune response.

Apoptosis plays a critical role in the development and maintenance ofthe immune system (35-37). Members of the tumor necrosis factor (TNF)family can regulate apoptosis in addition to an array of otherbiological effects such as cell proliferation and differentiation (38).Despite the functional redundancy of this family, specificity may beaccomplished by coordinating the spatial and temporal expression ofTNF-related ligands and their receptors and by restricting theexpression of signal transduction molecules to specific cell types. TNFreceptors interact with a family of molecules called TRAFs (TNFreceptor-associated factors)¹ that act as adaptors for downstreamsignaling events (39). For example, TRAF2 activates NF-κB (40) and alsoc-Jun N-terminal kinase (JNK) (41-43). The biochemical events leading toapoptosis involve the caspase family of cysteine proteases (44), whereasNF-κB appears to inhibit cell death (45). The TNF receptor family canalso regulate apoptosis by modulating the expression of Bcl-2 andBcl-2-related proteins (46, 47). Recent data indicates that the Bclfamily controls apoptosis by altering transmembrane conductance inmitochondria and by preventing the activation of caspases (48-50). Animportant role of TNF members in dendritic cells (DC) biology hasrecently emerged. DCs have several specializations that lead to thestimulation of naive T cells and play a role in the initiation of theimmune response (51). TNF-α and CD40L are molecules involved in thedifferentiation of DC from CD34⁺ bone-marrow or cord blood progenitors(52-54). Moreover, CD40L increases DC survival, upregulates MHC andcostimulatory molecule expression and induces the expression of avariety of cytokines (e.g., IL-12) in DC (55). Both CD40 and TNFRinteract with TRAF2, suggesting that TRAF2 plays a role in DC function.

Recently, TRANCE (TNF-related activation-induced cytokine), a novelligand of the TNF family was cloned during a search for apoptosisregulatory genes. Remarkably, and unexpectedly, TRANCE expression isrestricted to lymphoid specific organs and is selectively expressed in Tcells (56). Disclosed herein is the discovery that TRANCE-R signals viaTRAF2 in thymocytes and increases DC survival by upregulating Bcl-x_(L)expression, a property shared with CD40L. However, unlike CD40L, TRANCEselectively acts on mature DC but not on B cells. In addition, highlevels of the TRANCE-R are only detected on DC suggesting that a majorfunction of TRANCE in vivo is to modulate DC activity. Hence, TRANCE canalso modulate T cell activation and immune response to an antigen.

Materials and Methods

Expression and Purification of Soluble TRANCE. A FLAG epitope-taggedTRANCE molecule (FLAG-TRANCE) was expressed in 293T cells and purifiedas described (56). To create a human CD8-TRANCE recombinant molecule(hCD8-TRANCE), the extracellular domain of murine TRANCE (a.a. 245-316of FIG. 4 (SEQ ID NO:4)) was fused to human CD8α (a.a. 1-182) andproduced in a baculovirus expression system according to themanufacturer's instructions (BaculoGold, Pharmingen, Dan Diego. CA).hCD8-TRANCE was purified on CNBR-activated Sepharaose gel conjugated toOKT8 following the manufacturer's protocol (Pharmacia Biotech,Piscataway, N.J.). mCD8-CD40L in insect cell culture supernatant waskindly provided by Dr. Randolph J. Noelle (Dartmouth Medical School,Hanover, N.H.).

Mice. C57BL/6 (H-2^(b)) and BALB/c (H-2^(d)) mice were from TaconicFarms (Germantown, N.Y.). Transgenic mice expressing a dominant negativeform of TRAF2 (TRAF2.DN) were engineered as described (57).

Cells. Bone-marrow derived DC (BMDC) were generated as described (58)and were used on day 8 of culture. Enriched populations of fresh lymphnode or splenic DC were prepared by digesting organs with collagenasethen selecting for low density cells via centrifugation on a Nycodenzcolumn (14.5% w/v in PBS+5 mM EDTA; Nycomed, Oslo, Norway) for 15 min.at 4° C. Mature spleen DC were prepared by culturing freshly isolatedspleen DC overnight as described (59). The cytokine-induced generationof human DC from PBMCs was performed as described (60). After 2 days inmonocyte conditioned medium (MCM), TRANCE or PBS was added to the DCs.Lymph node T cells (99% CD3⁺ as assessed by flow cytometry) wereprepared by magnetic bead depletion (Dynal, Oslo, Norway) of class II,B220, NK1.1, and F4/80 positive cells. B cells were prepared by magneticdepletion of Thy1.2 positive cells (Dynal). Cell viability was assayedby trypan blue exclusion or by propidium iodide uptake.

Flow cytometry. DC phenotype was assessed by flow cytometry as described(61) using the following FITC or PE-conjugated mAbs: H-2K^(b), I-A^(b),ICAM-1, CD11b, CD11c, CD80, CD86, CD25, CD40 (PharMingen). Other mAbsused were biotinylated α-Fas, CD3-FITC, B220-FITC (PharMingen) andNLDC-145-FITC. The expression of TRANCE-R was assessed using thehCD8-TRANCE fusion molecule at 10 μg/mL at 4° C. followed bybiotinylated OKT8 mAb and then streptavidin-PE (BioSource International,Camarillo, Calif.). Negative controls were performed by omittinghCD8-TRANCE. For analysis of TRANCE-R expression on resting B cells andfresh DC, low density cells were stained with FITC-B220 or FITC-CD11c,respectively and analyzed on a “FACSCAN” (Becton Dickinson, MountainView, Calif.).

Mixed leukocyte reaction. BMDC treated for 48 hours in the presence orabsence of recombinant TRANCE were cultured with 1×10⁵ purifiedallogeneic T-cells in flat bottom 96 well plates in a final volume of200 μl for 3 days and then pulsed for eight hours with 0.5 μCi of³[H]-Thymidine (Dupont NEN®, Boston, Mass.). The cells were thenharvested on glass fiber filters and ³[H]-Thymidine incorporation wasmeasured using a standard scintillation-detection procedure.

c-Jun N-Terminal Kinase Assays. 2-5×10⁶ cells from TRAF2.DN transgenicmice or from control littermates were incubated 1-2 hours at 37° C. onplates coated with OKT8 antibody (10 μg/mL). The cells were treated witheither soluble TRANCE or an equal volume of PBS before harvesting at theindicated time points and frozen in a dry ice/ethanol bath. INK wasimmunoprecipitated with α-JNK1 antibody (Santa Cruz Biotechnology, SantaCruz, Calif.) and kinase activity assessed as described (56).

Western and RT-PCR analysis of Bcl-x_(L) and Bcl-2. BMDC (8×10⁶/well)were cultured in RPMI in six well plates and treated with PBS,FLAG-TRANCE (1 μg/mL) or soluble CD40L for 0 or 24 hours. The cells werelysed, and 50 μg of protein from each sample were resolved on a 12%SDS-PAGE gel and transferred to Immobilon-P membranes (Millipore,Bedford, Mass.). The blots were blocked in 5% skim milk, probed withα-Bcl-2 (4C11) or α-Bcl-x_(L) (236) [Kindly provided by Dr. GabrielNunez, Univ. of Michigan] and detected with the appropriateHRP-conjugated secondary antibodies and enhanced chemiluminesencesubstrate (ECL, Amersham Corp., Arlington Heights, Ill.). For RT-PCRanalysis of bcl-x_(L) mRNA expression, BMDC (2×10⁶ cells/well) werecultured in 24 well plates, treated with the appropriate reagents andquickly frozen in a cry ice/ethanol bath at the various time points.Total RNA was extracted (RNEasy, Qiagen Inc., Chatsworth, Calif.) andcDNA was diluted to allow PCR amplification to occur as a linearfunction of starting concentrations. PCR was performed using theconditions and primers as described (47).

Results and Discussion

TRANCE-R is expressed at high levels in dendritic cells. To identifycells that express TRANCE-R, hCD8-TRANCE was used as a molecular probefor FACS analysis. TRANCE-R was detected on mature BMDC, freshlyisolated lymph node DC and freshly isolated spleen DC (FIG. 5). TRANCE-Rwas greatly upregulated upon the maturation of spleen DC induced byovernight culture. No expression could be detected on freshly isolatedlymph node B cells, lymph node T cells, thymocytes or peritonealmacrophages. Therefore, the highest levels of TRANCE-R expression arefound on mature DC and suggest that the major role of TRANCE isrestricted to DCs.

TRANCE is a dendritic cell survival factor. The biological effects ofTRANCE were further studied on mature DCs. TRANCE-treated DCs formeddensely packed clusters while control, untreated cells exhibitedrelatively sparse aggregates (FIG. 10A). In addition, mature BMDCtreated with FLAG-TRANCE were significantly protected from spontaneouscell death compared to untreated cells. This effect was dependent on thedose of TRANCE (FIG. 10B). hCD8-TRANCE elicited similar results. Thiseffect was not due to increased cell proliferation since the totalnumber of cells remained the same over time. TRANCE significantlyprevented DC cell death until day 6, whereas untreated cells were almostcompletely dead by day 3 (FIG. 10C). A similar effect on DC survival wasobserved with human monocyte-derived DC (FIG. 10D). Confirming previousdata CD40 ligand (CD40L) also induced the clustering of DC (62, 63) andenhanced DC survival comparably to TRANCE (FIG. 10C).

CD40L upregulates the anti-apoptotic molecule, Bcl-x_(L), in B cells andprotects them from Ig-receptor mediated cell death (47). In addition,CD40L upregulates Bcl-2 in human DC derived from CD34⁺ progenitor cells,a phenomenon which was correlated with a resistance to Fas mediatedapoptosis (46). To determine whether TRANCE can influence Bcl-2 orBcl-x_(L) their expression in DC stimulated with TRANCE or CD40L wasmeasured by Western analysis. BMDC expressed relatively high levels ofBcl-2 and relatively low levels of Bcl-x_(L) after reaching maturity inGM-CSF (FIG. 10E, 0 hr). FLAG-TRANCE and CD40L stimulation lead toincreased Bcl-x_(L) expression by 24 hours. Bcl-x_(L) expression wasnearly absent in cells treated with medium alone bcl-x_(L) mRNA wasupregulated in TRANCE-treated DCs suggesting a transcriptional asopposed to post-transcriptional regulation. In contrast Bcl-2 levelswere decreased in both the TRANCE-treated and untreated cells (FIG.10E). These results suggest that TRANCE, in addition to CD40L,upregulates Bcl-x_(L) in DC which enhances their viability in vitro.

TRANCE enhances DC-mediated T cell proliferation. To examine thefunctional consequences of TRANCE on DCs the MLR stimulating ability ofDC treated with TRANCE was measured. Increasing doses of FLAG-TRANCEenhanced DC survival at 48 hours which, in turn, led to a proportionalincrease in the stimulation of T cell proliferation (FIG. 11A). Whenequivalent numbers of viable TRANCE-treated or untreated DC were used inan MLR, there were no differences in T cell proliferation, suggestingthat changes in the expression of costimulatory and antigen presentingmolecules did not account for the enhanced T cell proliferation (FIG.11B). To verify this, the levels of several surface markers were testedby FACS to evaluate any TRANCE-mediated changes to the DC phenotype.There was a slight but reproducible downregulation of MHC II expressionand a slight upregulation of MHC I expression (FIG. 11C). There were noTRANCE-mediated perturbations in the expression of the costimulatorymolecules CD80 (B7-1) or CD86 (B7-2), and no changes in the expressionof the adhesion molecules ICAM1, CD11b and CD11c. Interestingly, CD40expression increased but Fas or TRANCE-R did not. In sum, TRANCEenhances DC mediated T cell proliferation by increasing the survival ofDCs.

TRANCE does not affect B cell proliferation. Expression of high levelsof the TRANCE-R appeared restricted to DC by FACS analysis. However, itwas found that TRANCE could activate JNK in thymocytes (56) suggestingthat FACS analysis might lack the sensitivity to detect low levels ofreceptor. To further examine the specificity of TRANCE for DCs, itsability to induce B cell proliferation or survival was tested, twofunctions mediated by CD40L. Recombinant hCD8-TRANCE, tested for itsanti-apoptotic function in BMDC, could not stimulate B cellproliferation (FIG. 12) nor could it activate JNK activation (56). Incontrast, CD40L efficiently stimulated B cell proliferation in a dosedependent manner (FIG. 13). Finally, TRANCE could not prevent thespontaneous apoptosis of B cells and T cells as assessed by propidiumiodide uptake. Therefore, functionally, TRANCE appears to exhibitdifferent cellular specificities and functions when compared to CD40L.

TRANCE-mediated JNK induction requires functional TRAF2. Recruitment ofTRAF2 to the TNFR complex or the CD40 receptor complex is necessary forINK activation (41-43, 57). To test the possibility that TRANCE-R alsosignals via TRAF2, TRANCE-mediated INK activation was analyzed inthymocytes from transgenic mice over expressing a dominant negative formof TRAF2 (TRAF2.DN) (57). INK activity peaked 2.5 fold over unstimulatedcells at 5 minutes in control littermates whereas INK induction wassignificantly reduced in TRAF2.DN thymocytes (FIG. 9). These resultssuggest that signaling from the TRANCE-R requires TRAF2. TRANCE-mediatedINK induction in DC could not be assayed since TRAF2.DN expression hasbeen restricted to lymphocytes in the TRAF2.DN transgenic mice. Inaddition, INK activity was constitutively high in mature DC (56), whichare also known to have high levels of activated NF-κB (64), thusconfounding detection of increased JNK activity.

It has been demonstrated that TRANCE, in addition to CD40L, is amodulator of DC function. Similar to CD40L, TRANCE modulates thesurvival of mature, DC by regulating the expression of Bcl-x_(L). Incontrast to CD40L, however, TRANCE does not act on other APC such as Bcells. The signal transduction pathways downstream of TRANCE-R thatregulate DC activities remain unknown. TRANCE appears to signal viaTRAF2, at least in thymocytes, suggesting that TRAF2 may play a criticalrole in mediating signals for differentiation, activation and survivalin DC.

These findings complement the description of the selective expression ofthis new TNF family member in T cells. The high level of expression ofTRANCE-R on DC suggests a specific role for TRANCE in T cell-DCcommunication during the primary immune response. Rapid upregulation ofTRANCE upon TCR engagement on T cells (56) could specifically enhancethe survival of DC during antigen presentation. Both antigen-specific Tcells and the antigen presenting DC would therefore depend on each otherfor activation and survival, respectively. Mature DC which fail topresent antigen to T cells would not receive T cell help and thereby dieof neglect. This T cell-DC interaction is likely to occur in the T cellarea of lymphoid organs which contain DC of mature phenotypes (65). DCcan only be detected in afferent lymph not efferent lymph suggestingthat DCs are destined to die when they migrate to the lymph node. Thus,TRANCE is important to maintain DC survival. Furthermore, DC pulsed exvivo with an antigen and TRANCE, and then reintroduced into an animalcan be used to induce immunity to tumor or viral antigens in vivo (66).Consequently, TRANCE is a tool to specifically enhance DC function byenhancing their survival in vivo.

Example III Methods for Modulating Immune Response to an Antigen in anAnimal

Dendritic cells are a specialized class of leukocytes found in manytissues and most abundantly in the T-dependent areas of lymphoid organs.As explained above, they play a fundamental role in antigen presentationto T-cells, being very potent since only small numbers of dendriticcells are sufficient to induce T-cell responses. Dendritic cells canprime naive T-cells (CD4 and CD8) both in vitro and in vivo. The antigenpresenting function of dendritic cells is related to their highexpression of both class I and class II MHC products, as well asdifferent costimulatory and adhesion molecules. Moreover, dendriticcells have special antigen handling mechanisms, including thelectin-type DEC-205 in mice and the mannose receptor in human andabundant MHC class II rich vacuoles. Mature dendritic cells areshort-lived in vitro. Different TNF family members appear to promotedendritic cell survival. This is the case for CD40L, TNFα and morerecently TRANCE, all products of activated T-cells. However, heretofore,the effect of increasing dendritic survival on in vivo immune responseis not known. Set forth herein are experiments designed to determine theeffect of immune response to an antigen in vivo wherein dendritic cellsare pulsed ex vivo with TRANCE, conservative variants or fragmentsthereof, analogs or derivatives thereof, or fusion proteins comprisingTRANCE, conservative variants thereof or fragments thereof, and anantigen, and then are reintroduced into an animal. Also set forth hereinare the results of such experiments.

Methods

Animals: C57BL/6 (H-2^(b)) female mice 6-8 weeks old were used in thisstudy and were from Taconic Farms (Germantown, N.Y.).

Dendritic cells: Bone marrow derived dendritic cells were prepared aspreviously described by Inaba et al. On day 6 of culture immaturedendritic cells were pulsed for 6 hours with Purified Protein Derivative(PPD) of Mycobacterium Tuberculosa at 10 mg/ml and the replated in 100mm culture dishes in order to induce maturation. On day 7 dendriticcells were pulsed with fusion protein mTRANCE-hCD8 (1 mg/ml) or anequivalent volume of PBS. On day 8 mature dendritic cells were washed 4times in Hank's buffer and resuspended at 4×10⁶/ml in the same buffer.

Priming with antigen-pulsed Dendritic Cells: 2×10⁵ (50 μl) dendriticcells were injected in the hind foot pad of C57Bl/6 mice.

Proliferation assays: Draining lymph nodes (popliteal, inguinal),mesenteric lymph node, and spleen were harvested from immunized mice andcell suspensions were prepared. Cells were cultured in flat-bottomed96-well plated, 3×10⁵ cells/well in 200 μl in the presence of increasingamount of PPD. Culture medium consisted of Click's medium supplementedwith 0.5% heat inactivated normal mouse serum, 10 mM Hepes, 2 mML-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 5×10⁴ M2-ME. On day 3, cells were pulsed for eight hours with 0.5 μCi of³[H]-Thymidine, then harvested on glass fiber filters and ³[H]-Thymidineincorporation was measured using a standard scintillation-detectionprocedure.

Results

As shown in FIG. 14, the proliferative response to PPD is surprisingly5-8 times higher in draining lymph nodes of mice which have receivedPPD+TRANCE pulsed dendritic cells, than of mice which have receivedPPD-pulsed dendritic cells. Similar enhancement was achieved when DCswere treated with agonistic CD40L. The major difference was on day 6,which has been shown to correspond to the peak of proliferative responseafter priming with ex vivo antigen pulsed dendritic cells. However, asignificant increase in in vitro response to PPD was still observed 2weeks after the injection, which indicates the secondary response isalso increased. As expected, no significant proliferative response wasobserved in mesenteric lymph node. A significant response to PPD wasobserved in spleen of injected with PPD−+ TRANCE pulsed dendritic cells,but not with PPD-pulsed dendritic cells. Although Applicants are underno obligation to explain such results, and do not intend to be bound bysuch an explanation, Applicants have postulated that this result isrelated to the recirculation of PPD-specific memory T-cells through thespleen at that time.

As shown in FIG. 14, treatment of DCs ex vivo with TRANCE or CD40Lenhances the longevity of DCs in vivo as well as in vitro, which leadsto the enhanced immunogenicity of antigens (e.g., PPD) delivered by thetreated DCs.

CONCLUSION

Increasing survival of dendritic cells ex vivo before injection bypulsing them with TRANCE, conservative variants thereof; fragmentsthereof; analogs or derivatives thereof, or fusion proteins comprisingTRANCE, conservative variants thereof, or fragments thereof candramatically improve the adjuvant effect of dendritic cells in vivoregarding a particular antigen, which was also used to pulse thedendritic cells were ex vivo. In addition to the survival effect ofTRANCE on the dendritic cells, ex vivo exposure of the cells to TRANCEalso stimulates their production of cytokines, such as IL-12, forexample. Hence, dendritic cells treated in accordance with the method ofthe invention can be used as “natural adjuvant” in vivo for inducingefficient immune response to a bacterial, viral or tumor antigen inrodents, e.g., such dendritic cells have applications in humans toinduce immune response to viral or tumor antigens. Indeed, large numbersof human dendritic cells can now be generated from peripheral blood orbone marrow progenitors using cytokines cocktails. Furthermore,fragments of TRANCE, conserved variants thereof, and even fusionproteins comprising TRANCE, such as mTRANCE-hCD8 described herein,increase in vitro survival of monocyte-derived human dendritic cells. Asresult, TRANCE or CD40L have applications in immunotherapy as a specifictool to increase in vivo dendritic survival in humans.

Example IV The TNF-SuperFamily Member TRANCE, is DifferentiallyExpressed on T Cell Subsets and Induces Cytokine Production in DendriticCells

As explained in the above Examples, TNF and TNF receptor family ofproteins play critical roles in the initiation and regulation of theimmune response. These proteins enable complex dialogue to occur betweencells within the immune system and with cells of other tissues. Despitethe apparent redundancy of the TNF/TNFR family as evidenced by thecontinually growing number of discovered ligand/receptor pairs and bythe common signaling transducers utilized by the receptors, the specificfunction of members of this family clearly exists as shown bygene-knockout studies, in which the deletion of one family member cannotby fully compensated by the others. Specificity may be achieved byrestricting their expression to particular cells and/or by linking theirsignal transducing effectors to cell-specific signaling pathways.

T-cells can modulate the function of dendritic cells (DC),antigen-presenting cells specialized in the activation of naive T-cells(67), via TNF-related molecules. CD40L, a CD4⁺ T-cell restrictedmolecule, has been shown to induce differentiation, cytokine production(TNF-α, IL-8, IL-12 and MIPα) and protection from spontaneous apoptosisin DC. TNF was also shown to enhance dendritic cell survival in vitro(63). IL-12-producing DC were shown to skew the response of T-cellstowards the Th1 phenotype (70, 71) suggesting that CD4⁺ T-cells expressCD40L to adjust the type of response (Th1 vs. Th2) by controlling DCfunction.

Further, Applicants have discovered and set forth in the above Examplesthat unexpectedly, TRANCE (TNF-related activation induced cytokine), amember of the TNF family (56) also called RANK-L (Receptor activatingNF-kB-ligand (72)) is a DC survival factor that regulates the expressionof the anti-apoptotic molecule, Bcl-x_(L) (73). TRANCE expressionappears restricted to T-cells whereas high levels of TRANCE-R areexpressed on mature DC. Hence, TRANCE/TRANCE-R interactions are involvedwith T-cell-DC communication (56, 72, 73). Furthermore, Applicants havediscovered that TRANCE expression has also been detected on osteoblastsand was shown to be required for osteoclast differentiation from myeloidprogenitors (74, 75). In addition, a soluble decoy receptor (OPG/OCIF)for TRANCE can block TRANCE-mediated osteoclast differentiation, andthus modulate T-cell-DC interactions (76).

Applicants set forth herein the discovery that CD4+ and CD8+ T-cells,when activated through the TCR/CD3 complex, express high level of TRANCEand its expression is strongly enhanced by CD28-mediated costimulationon CD4+ T-cells. In addition, Applicants have discovered that TRANCE hasno significant effects on activated T and B cells although they canexpress low level of TRANCE-R when activated. TRANCE can upregulate bothproinflammatory cytokines and factors in DCs that mediate T-cell growthand differentiation, a property shared with CD40L. Moreover, Applicantshave discovered that, surprisingly and unexpectedly, TRANCE cooperateswith a protein of the TNF family, such as CD40L or TNF-α to enhance thesurvival of DCs. Therefore, TRANCE plays an important role in theregulation of T cell responses by controlling the lymphocyte-stimulatorycapacity of DC.

Materials and Methods

Expression and Purification of Soluble TRANCE-R-Fc and hCD8-TRANCE. Tocreate a TRANCE-R-Fc recombinant molecule (TR-Fc), the Fc portion ofhuman IgG1 was fused to the C-terminal end of the extracellular domainof the murine TRANCE-R (also called RANK (11)) and produced in abaculovirus expression system according to the manufacturer'sinstructions (BaculoGold, Pharmingen, Dan Diego. CA). TR-Fc was purifiedfrom the culture supernatants on protein A sepharose bead. (PharmaciaBiotech, Piscataway, N.J.). hCD8-TRANCE was prepared as previouslydescribed (73).

Determination of the specificity of hCD8-TRANCE and TR-Fc. 293T cellsgrown in DMEM 10% FCS were transfected with expression vectorscontaining mTRANCE cDNA, mTRANCE-R or mFas cDNA by calcium phosphateprecipitation. Cells were incubated with 10 mg/ml of hCD8-TRANCE or 5mg/ml of TR-Fc and binding was revealed by FACS as described below.

Mice. C57BL/6 (H-2b) and BALB/c)(H-2^(d)) mice were from Taconic Farms(Germantown, N.Y.).

Medium. The culture medium used was RPMI 1640 supplemented withheat-inactivated 5 FCS, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/mlstreptomycin, 10 mM HEPES and 5×10-5 M 2-ME.

Cells. Mature bone-marrow derived DC (BMDC)3 were generated as describedand were used on day 8 of culture. Splenic DC were isolated as described(59) and cultured overnight to induce maturation. Lymph node T-cells(≧99% CD3⁺ as assessed by flow cytometry) were prepared by magnetic beaddepletion (Dynal, Oslo, Norway) of class II, B220, NK1.1, and F4/80positive cells. Th1 and Th2 clones derived from DO11.10 TCR transgenicmice, were tested by intracellular staining of IL-4, IL-10 and IFN-γ.The Th1 clones were IL-4⁻ IL-10⁻ IFNγ⁺ and the Th2 clones were IL-4⁺IL-10⁺ IFNγ⁻. B cells (≧94% B220⁺) were prepared from spleen cells bymagnetic bead depletion of Thy-1.2 positive cells (Dynal). Cellviability was assayed by trypan blue exclusion or by propidium iodideuptake.

Flow cytometry. The expression of TRANCE on activated CD4+ and CD8+T-cells was assessed using the TR-Fc fusion protein at 5 mg/ml followedby FITC-conjugated goat anti-human IgG (Fc specific) F(ab′)₂ fragment(Jackson. Laboratories, West Grove, Pa.). The negative control consistedof normal hIgG1 (Sigma, St Louis, Mich.). T-cell and thymocyte subsetswere sorted using a FACS “VANTAGE”, (Becton Dickinson, Mountain View,Calif.).

RT-PCR analysis. For semi-quantitative PCR analysis total RNA wasextracted from FACS sorted T-cell subsets (RNA Isolation Kit,Stratagene, Calif.) cultured in 24 well plates coated with or withoutanti-CD3 (145-2C11, 10 mg/ml) for 3.5 h and subjected to RT-PCR aspreviously described (56). HPRT: (sense: 5′-GTA ATG ATC AGT CAA CGG GGGAC-3′ (SEQ ID NO:15), antisense: 5′-CCA GCA AGC TTG CAA CCT TAA CCA-3′(SEQ ID NO:16)). TRANCE: (sense: 5′-CCT GAG ACT CCA TGA AAA CGC-3′ (SEQID NO:11), antisense: 5′-TAA CCC TTA GTT TTC CGT TGC-3′ (SEQ ID NO:12)).CD40L: (sense: 5′-GTG GCA ACT GGA CTT CCA GCG-3′ (SEQ ID NO:17),antisense: 5′-GCG TTG ACT CGA AGG CTC CCG-3′ (SEQ ID NO:18)). The PCRproducts were analyzed by Southern blot as previously described (56).

Ribonuclease protection assays. Total RNA was obtained from 1×10⁷ BMDC(RNA Isolation Kit; Stratagene, Calif.) treated for 12 h withhCD8-TRANCE (1-5 mg/till), a 1:100 dilution of mCD8-CD40L baculoviralsupernatants or with an equivalent volume of PBS. 5 mg of RNA from eachsample was hybridized to a P³²-labeled antisense RNA probe set (mCK-1,mCK-2, mCK-3, mAPO-2; Pharmingen, Calif.), digested with RNAse+T1nuclease and the protected probe fragments were resolved on 5%polyacrylamide gels following the manufacturer's protocols. Bandintensity was quantified by phosphorimaging (Molecular Imager System;BioRad, CA) and normalized to the intensity of the GAPDH probe.

Results

The regulation of TRANCE mRNA expression in T cells. TRANCE mRNAexpression was measured in sorted naive (CD44^(low)) and memory(CD44^(high)) LN T-cell subsets and in various thymocyte populations(FIG. 17). Purifed T-cells and thymocytes were stimulated with anti-CD3mAb or left unstimulated for 3.5 hr and levels of TRANCE mRNA wereassessed by semi-quantitative RT-PCR analysis (FIG. 17). Resting CD8⁺and CD4⁺ memory cells expressed high levels of TRANCE whereas restingnaive CD8⁺ and CD4⁺ T-cells did not express TRANCE mRNA. UponCD3-stimulation all T-cell subsets upregulated TRANCE with the highestlevels observed in CD3-stimulated memory CD4⁺ and CD8⁺ T-cell subsets.CD40L mRNA expression was also examined and shown to be upregulated inactivated CD4⁺ naive and memory T-cells (77). In contrast, CD40L mRNAexpression was very weak in CD8⁺ T-cells (FIG. 17).

Although TRANCE was not expressed in resting peripheral T-cells,substantial levels of TRANCE mRNA were detected in SP CD4+CD8⁻ andCD4⁻CD8⁺ thymocytes. In contrast, CD40L was restricted to CD4⁺CD8⁻thymocytes (FIG. 17). Hence, TRANCE and CD40L are transiently expressedupon maturation of thymocytes (78).

The regulation of TRANCE protein expression on the surface of T cells.TR-Fc fusion protein that could specifically recognizeTRANCE-transfected 293T cells but not 293T cells transfected with vectoralone (FIG. 18A) was used to detect surface TRANCE expression onT-cells. TRANCE was not detected on resting CD4+ or CD8⁺ T-cells (FIG.19). On CD4⁺ T-cells, surface TRANCE expression was detected as soon as4 h after anti-CD3 and anti-CD28 stimulation, peaked around 48 h andremained high at least until 96 hours (FIG. 19). The kinetics of TRANCEexpression on CD8⁺ T-cells were slower than that on CD4⁺ T-cells andCD8⁺ T cells expressed lower levels of TRANCE than CD4⁺ T whenstimulated with anti-CD3 and anti-CD28 mAbs (FIGS. 20 and 21). However,CD4⁺ and CD8⁺ T cells stimulated with anti-CD3 in the absence ofcostimulation expressed similar low levels of TRANCE (FIG. 21). Indeed,anti-CD28 mAb-mediated costimulation greatly enhanced TRANCE expressionon CD4⁺ but not significantly on CD8⁺ T cells (FIG. 20). To determinewhether TRANCE expression is restricted to T helper subsets, Th1 and Th2clones derived from DO11.10 TCR transgenic mice were stained with TR-Fc.As shown in FIG. 21, TRANCE was not detected on resting clones but wasstrongly upregulated on both Th1 and Th2 clones after anti-CD3stimulation although the Th1 clones consistently expressed higher levelsthan the Th2 clones.

In order to further analyze the regulation of TRANCE expression onactivated T cells, the effects of several cytokines were tested.Purified T cells were stimulated for 60 hours in the presence or in theabsence of cytokines. Among the different cytokines tested, it wasdiscovered that IL-4 (20 ng/ml) significantly inhibited the expressionof TRANCE on activated CD4+ but not CD8⁺ T cells (FIG. 22). In contrast,TGF-β1 (1 ng/ml), IFN-α (1000 U/ml), IFN-α (100 Md.), IL-2 (50 U/ml),TNF-α (50 ng/ml) or LT-α (50 ng/ml) had no significant effects on TRANCEexpression.

TRANCE-receptor is expressed on activated T and B cells. It has been setforth that levels of TRANCE-R are expressed on mature DC (73). SinceTRANCE-R has also been detected on activated human T cells (72) andTRANCE can activate c-Jun N-terminal kinase in thymocytes (73),expression of TRANCE-R was analyzed on murine T cells using thehCD8-mTRANCE fusion molecule (FIG. 18B) and FACS analysis (FIG. 23). Asexplained in Examples set forth above, resting T-cells did not show anydetectable TRANCE-R expression on their surfaces (FIG. 23A). However,when T cells were stimulated with anti-CD3, low levels of TRANCE-R weredetected only after 48 h of simulation and were not further increased byanti-CD28-mediated costimulation. TRANCE-R expression was not enhancedby IL-4 and/or TGF-b1, either (FIG. 23A) despite a previous studyshowing that these cytokines enhance the expression of TRANCE-R onactivated human T cells (72). In addition, Applicants have discoveredthat TRANCE did not have any effect on the survival or primary orsecondary proliferative responses of murine CD4+ or CD8⁺ T cells despitesignificant TRANCE-R expression on those cells. TRANCE-R expression canbe also detected on activated B cells (FIG. 23B). TRANCE-R expressionwas detected after 24 h of stimulation and peaked at 48 h. Moreover,TRANCE-R expression was significantly enhanced by CD40 cross linking onB cells but only slightly by anti-μ+IL-4. This stimulatory requirementsof TRANCE-R expression on B cells was similar to that of Fas expression(FIG. 23B). The level of expression of TRANCE-R on mature DCs wasconsistently at least 10-fold higher than on activated B cells. TRANCEhad no effect on proliferation, the expression of surfaceactivation/adhesion markers or survival of B cells stimulated to expressTRANCE-R.

TRANCE induces cytokine production in DC. TRANCE and CD40L canupregulate Bcl-x_(L) expression and protect DC against spontaneousapoptosis in vitro (73). In addition to its survival-enhancing functionin DC, CD40L can induce IL-12 and IL-18 expression (80) which in turncan promote a Th1-mediated immune response, and an array of cytokinesinvolved in T-cell activation (IL-1, IL-6, IL-15, TNF-α) (80-81). Todetermine if TRANCE plays a similar role in cytokine regulation,TRANCE-treated or PBS-treated DC were subjected to ribonucleaseprotection assays (RPA) with probes specific for a variety of knowncytokines (FIG. 24). Applicants have discovered that TRANCE induces theexpression of the proinflammatory cytokines IL-1β, IL-1Ra, IL-6, theT-cell and NK cell activating cytokine, IL-15 (FIG. 24). TRANCE alsoupregulates the mRNA encoding the p40 subunit of IL-12. In this assay,IL-12 p35 mRNA was not detected. Although under no duty to explain suchlack of detection, and certainly not intending to be bound by anyhypothesis presented here, it is hypothesized that the lack of detectionof IL-12 p35 mRNA was probably because the steady state level of p35mRNA was below the limit of detection. TRANCE had no apparent effect onthe expression of IL-2, IL-4, IL-5, IL-9, IL-10, IL-1α, TNF-α, TNF-β(LT-α), LT-β, IFN-γ or IFN-β (FIG. 24). By the same method, it was shownthat CD40L also upregulated the expression of IL-1β, IL-1Ra, IL-6, IL-12p40 (but not p35), IL-15. However, TRANCE and CD40L differed in theregulation of TGF-β2 expression; TRANCE induced TGF-β2 expression anddown-regulated TGF-β1 (FIG. 24) whereas CD40L upregulated both TGF-β1and TGF-β2.

TRANCE cooperates with a protein of the TNF family to enhance thesurvival of DC. Applicants have discovered that, surprisingly andunexpectedly, during a T-cell-DC interaction both TRANCE and a proteinof the TNF family, such as CD40L or TNF-α cooperatively enhance DCsurvival. As shown in FIG. 16, the addition of both ligands together,inhibited cell death to a greater degree than either ligand alone (FIG.16). TNF could also prevent spontaneous apoptosis as previouslydescribed (82) and also cooperates with TRANCE to enhance splenic DCsurvival (FIG. 16). GM-CSF, a cytokine required for DC differentiation,had little effect on splenic DC survival, however, its effect wassignificantly amplified when administered with TRANCE (FIG. 16). Thecooperative effect of TRANCE, CD40L and TNFα on DC survival was alsoobserved with BMDC (FIG. 16).

Discussion

The data presented here further uncovers the role of TRANCE/TRANCE-R inthe immune system. Previously, TRANCE was shown to enhance the survivalof DCs, a property shared with CD40L (21, 73). However, heretofore, thecooperative nature of such proteins in increasing survival of dendriticcells was not known. It has been set forth above that TRANCE isexpressed on both activated CD4+ and CD8⁺ T-cells, with higher levels ofexpression observed on CD4⁺ T cells, while CD40L is expressed only inactivated CD4⁺ cells. Hence, TRANCE allows CD4⁺ T-cells to modulate DCfunction independently of CD40L. TCR stimulation by itself is sufficientto induce TRANCE expression on T cells, which can be further increasedby CD28-mediated costimulation on CD4+ T cells but not significantly onCD8+ T cells. In contrast, CD28 costimulation does not modify the levelbut only the kinetics of expression of CD40L on activated CD4+ T cell(83, 84). Moreover, the kinetics of TRANCE expression during CD4+ T cellactivation are different from those described for CD40L (85). Indeed,maximal levels of TRANCE expression are detected at 48 h afterstimulation and persist for at least 2 days more whereas CD40L proteinhas been shown to be rapidly expressed and then to wane within 16-24 h(85). Therefore, TRANCE acts at later time point than CD40L during CD4⁺T-cell mediated immune response to regulate the functions of DCs.Interestingly, CD40 is expressed on both immature and mature DC and cansignal DC maturation (67), whereas TRANCE-R is only expressed on matureDC (73).

Furthermore, it has been shown that TRANCE-R is not detected on restingT-cells by FACS analysis (73). In addition, just as with human activatedT-cells, TRANCE-R can be detected on murine T-cells when activated.However, TRANCE does not effect proliferation, costimulation, survivalor cell death in these cells, contrary to what has been observed inhuman T cells (72). Although under no duty to explain such adiscrepency, and certainly not intending to be bound by any hypothesisfor the cause of these discrepencies, these discrepancies could reflectfunctional differences between the human and mouse TRANCE-R in T-cellsand/or to differences in culture and stimulation conditions. Moreover,contrary to previous reports which disclose that a soluble form ofTRANCE can be shed from TRANCE-transfected 293 cells, TRANCE was notshed in vitro from activated T cell hybridoma. Hence, the relative lowlevel of TRANCE-R on activated T-cells is not due to the production ofsoluble TRANCE by those cells.

In addition, set forth herein is a discovery that activated B cells alsoexpress low levels of TRANCE-R. Similar to activated T-cells, theproliferation, the survival and the phenotype of activated B cells werenot affected by TRANCE. Hence, data set forth herein indicates that themajor immune target cells for TRANCE are DCs, which is a significant andimportant difference with CD40L. This is an important difference withCD40L, which has also a major effect on B cell function.

In addition to its ability to enhance DC survival, TRANCE also promotesthe production of various cytokines (e.g., IL-12, IL-15, IL-1 and IL-6)in DCs. CD40L is known to be a major stimulus inducing IL-12 productionby DC (25, 69), a critical cytokine involved in Th1 differentiation(71). However, neutralizing antibodies to CD40L fail to completely blockIL-12 production in an MLR with T-cells and DC (69) and CD40L knockoutmice are still able to produce IL-12 (87). TRANCE also induces IL-12production in DC. Consequently, as set forth herein, TRANCE complementsCD40L in vivo to promote DC-mediated Th1 differentiation. Interestingly,IL-4, which is required for Th-2 cell differentiation (88) substantiallyinhibits TRANCE expression on activated CD4+ T cells. Thus IL-4producing cells down regulate TRANCE expression on T cells during T cellpriming leading to a decreased IL-12 production by DC and thereforedecreased Th1 differentiation. Consistent with the potential role ofTRANCE in enhancing Th1 responses and the effect of IL-4 on TRANCEexpression are the lower levels of TRANCE on activated Th2 clones ascompared to the Th1 clones from DO11.10 mice.

IL-15 is a cytokine that shares functional similarities and receptorchain usage with IL-2 (89). It is a mitogen for NK cells (89) and is aT-cell growth factor (91) and chemoattractant (91). Similar to humanCD34+ derived DC, resting murine DC expressed very low levels of IL-15mRNA (80), which were dramatically upregulated upon TRANCE-R or CD40triggering. In addition, IL-15 can enhance the survival of activatedT-cells (92) and specifically activates memory CD8⁺ T cells (93). Hence,activated/memory Th cells which express high levels of TRANCE promotetheir own survival by interacting with DC and inducing IL-15 production.Furthermore, just as with CD40L (80), TRANCE unexpectedly can alsotrigger the production of proinflammatory cytokines such as IL-1 andIL-6 which can amplify the immune response initiated by DC. TRANCE andCD40L therefore behave similarly in their ability to enhance DC-mediatedlymphocyte activation.

Furthermore, it is clearly set forth herein that TRANCE and a protein ofthe TNF family, cooperate to enhance the survival of Dcs, and thus canbe used to modulate immune response in an animal, or to treat an immunesystem related disease or disorder in an animal. In particular, TRANCEand CD40L, both of which are expressed on CD4+ T cells, cooperate toenhance the survival of DCs. Hence, DC survival in vivo utilizes thecombined action of several TNF-family members, including TNF-α, whichare likely to be provided by activated CD4+ and CD8+ T-cells and thosepresent in the local microenvironment.

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Many other variations and modifications of the invention will beapparent to those skilled in the art without departing from the spiritand scope of the invention. The above embodiments are, therefore, merelyexemplary, and all such variations and modifications are intended to bewithin the scope of the invention as defined in the appended claims.

It is further understood that all base sizes or amino acid sizes, andall molecular weight or molecular weight mass values given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated antibody that is specific for a human form of TRANCE andnot a mouse form of TRANCE and binds to a human TRANCE polypeptide,wherein said polypeptide comprises the amino acid sequence of SEQ IDNO:2.
 2. The antibody of claim 1, wherein said antibody is a monoclonalantibody.
 3. The antibody of claim 1, wherein said antibody is apolyclonal antibody.
 4. The antibody of claim 1, wherein said antibodyis a chimeric antibody.
 5. The antibody of claim 1, wherein saidantibody is detectably labeled.
 6. The antibody of claim 5, wherein adetectable label is selected from the group consisting of alkalinephosphatase, peroxidase, and radioactive isotopes incorporated into theantibody.
 7. An isolated antibody that is specific for a human form ofTRANCE and not a mouse form of TRANCE and is produced from an immunogen,wherein the immunogen is a polypeptide comprising the amino acidsequence of SEQ ID NO:2.